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Labjournal Surchem August

Experiment 63e: Anti Mouse/Rabbit on PDITC

protocols used :Plasma activation, iRIf slide preparation

2015.08.29

Experiment/Protocol

3 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h
desiccator: 15 min
stored at 4 °C

2015.08.31

Experiment/Protocol

spotting pattern:
slides: 424, 012

#	spot	Concentration
1	GFP	0.5 mg/ml
2	anti HCV (rabbit)	0.5 mg/ml
3	anti Tetanus (mouse)	0.5 mg/ml

slide: 254

#	spot	Concentration
1	GFP	1 mg/ml
2	anti HCV (rabbit)	1 mg/ml
3	anti Tetanus (mouse)	1 mg/ml

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
anti- rabbit	6	30	600
Buffer	7	60	300	1x
anti- mouse	8	30	600
Buffer	9	60	300	1x

Results

Slide 012: Quotion picture

Slide 012: binding curves

Slide 424: Quotion picture

Slide 424: binding curves

Slide 254: Quotion picture

Slide 254: binding curves

→ the detection of GFP-, rabbit-, and mouse antibodies worked on all three slides

→ the increase in protein concentration of the spotted antigens on slide 254 does not lead to a increase of the detection signal → surface is with a protein concentration of 500 µg/ml already saturated

Experiment 63d: Stockholm II

protocols used :Plasma activation, iRIf slide preparation

2015.08.29

Experiment/Protocol

1 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h
desiccator: 15 min
stored at 4 °C

2015.08.30

Experiment/Protocol

spotting pattern:

slide-#:315

#	spot	Concentration
1-3	rhErbB2/Fc	100 µg/ml
4	GFP (desalt)	1 mg/ml
5	Tetanus antigene	100 µg/ml

2015.08.31

Experiment/Protocol

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
affibody	6	20	900	diluted 1:3
Buffer	7	60	300	1x
affibody	8	20	900	undiluted
Buffer	9	60	300	1x
Anti His	10	30	600	undiluted
Buffer	11	60	300	1x

Results

Slide 315: Quotion picture

Slide 315: binding curves

→ Binding of the anti-his antibody to the antigen (rhErbB2/Fc) was detected→ the (His tagged) antigen was immobilized on the surface

→ no binding of the affibody to its antigen couold be detected → either the affibody does not bind strong enough or the affibody is too small (~8 kDa) to generate a sufficient iRIf signal

Experiment 63c: Tetanus on PDITC

protocols used :Plasma activation, iRIf slide preparation

2015.08.29

Experiment/Protocol

2 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h
desiccator: 15 min
stored at 4 °C

2015.08.30

Experiment/Protocol

Spotting pattern:
Slides: 303,466

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	neg. con (Salmonella)	(1.24 mg/ml) desalt 1:3 diluted
3	Tetanus	(0.46 mg/ml) desalt undiluted

2015.08.31

Experiment/Protocol

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
Serum (-/+) Sig002	6	30	900	diluted in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

Slide 303: flushed with Serum (-) diluted 1:20 in BSA
Slide 466: flushed with Serum (-) diluted 1:30 in BSA

Results

serum (-) 1:20 dilution:

Slide 303: Quotion picture

Slide 303: binding curves

→ strong binding to the salmonella antigen was detected → SIG 002 could also experienced a previous salmonella infection. Signal overall was quite weak.

serum (-) 1:30 dilution:

Slide 466: Quotion picture

Slide 466: binding curves

→the 1:30 diluted serum(-) shows little unspecific binding → weak binding to tetanus antigen could be caused by low anti tetanus antibody titer from previous vaccination

→overall weak signal, reuslts rather inconsistent→ use new stock of blood serum for previous experiments

Experiment 63b: Ni-NTA iRIf slides

protocols used :Plasma activation, iRIf slide preparation

2015.08.29

Experiment/Protocol

4 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h
slides were sandwiched with NTA-solution and incubated o/n at 4 °C

2015.08.30

Experiment/Protocol

blocked slides with APTES blocking solution for 1 h
washed slides 2x with PBS for 10 min
incubated slides for 1 h in 1 % NiSO₄-solution
washed slides with PBS/2x diluted PBS
stored slides at 4 °C under N₂-atmosphere

Experiment 63a: Salmonella antigene on PDITC in iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.29

Experiment/Protocol

4 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h

spotting pattern:

slide-#: 208, 216, 423, 500

#	spot	Concentration
1	pos. control (GFP)	1 mg/ml
2	neg. control (mCherry)	1mg/ml
3	Salmonella Antigen (15)	undiluted

2015.08.30

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
slides were blocked with 10 mg/mL BSA in PBS for 30 min
slides were washed with PBS 3x 10 min
slides were washed with Aqua dest and dried

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
anti-Salmonella (1301, desalt)	6	30	900	1:2 Dilution in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

208: with PBS 423: with TBS 216: with TBS 500: with TBS (changed flush protocol: 1 ug/ml a-GFP; and used anti-Salmonella lysate 1:2 diluted in 5 mg/ml BSA in TBS)

Results

PBS:

Slide 208: Quotion picture

Slide 208: binding curves

→ only very weak Salmonella antigen/antibody binding detectable, probably due to old salmonella antigen and antibody stocks

TBS:

Slide 216: Quotion picture

Slide 216: binding curves

Slide 423: Quotion picture

Slide 423: binding curves

→ only very weak or no Salmonella antigen/antibody binding detectable, probably due to old salmonella antigen and antibody stocks

Salmonella Lysate:

Slide 500: Quotion picture

Slide 500: binding curves

→ much unspecific binding detected, probably due to binding of E. coli proteins of the salmonella antibody lysate to other E. coli proteins still present in the purified mCherry and salmonella antigen (both were expressed in E. coli)

Experiment 62c: PDITC/Ni-NTA slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.26

Experiment/Protocol

4 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 3 h
slides were stored under N₂-atmosphere at 4 °C

Experiment 62b:Tetanus antigen on PDITC slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.26

Experiment/Protocol

4 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 3 h
slides incubated o/n at 4 °C

2015.08.29

Experiment/Protocol

spotted Tetanus antigen

spotting pattern:

slide-#: 287, 024, 426, 443

#	spot	Concentration
1	pos. control (GFP)	1 mg/ml
2	neg. control (Salmonella antigen)	desalt 1:3
3	Tetanus antigen	undiluted

2015.08.30

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
slides were blocked with 10 mg/mL BSA in PBS for 30 min
slides were washed with PBS and 2x with 1/10 diluted PBS for 10 min
slides were dried and measured in iRIf

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
Serum (-/+) Sig002	6	30	900	1:10 diluted in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

Slides: 287, 024 (with Serum (-)) #287: Serum (-) was diluted only 1:10; changed to 1:30 because Serum(-) quite high Signal at negative control and Tetanus spot Flush protocol Slides: 426,443 (with Serum (+)) Flush protocol

Results

Serum (-):

Slide 024: Quotion picture

Slide 024: binding curves

Slide 287: ROI selection

Slide 287: binding curves

→ slide 024 shows low unspecific binding due to 1:30 dilution of the serum(-), slide 287 shows high unspecific binding due to the 1:10 dilution of the serum(-)

→overall signal much weaker than before, probably due to repeated freezing of the blood serum

Serum (+):

Slide 426: ROI selection

Slide 426: binding curves

Slide 433: ROI selection

Slide 433: binding curves

→slide 426 shows relativly weak unspecific binding and moderate binding of proteins to the Tetanus antigen, slide 433 has more unspecific binding

→overall signal much weaker than before, probably due to repeated freezing of the blood serum

Experiment 62a: PDITC/Ni-NTA slides for iRIf - Stockholm collaboration

protocols used :Plasma activation, iRIf slide preparation

2015.08.26

Experiment/Protocol

2 PDITC slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 3 h
slides were sancwiched with NTA-solution and incubated o/n at 4 °C

2015.08.27

Experiment/Protocol

blocked slides with APTES blocking solution for 1 h
washed slides 2x with PBS for 10 min
incubated slides for 1 h in 1 % NiSO₄-solution
washed slides with PBS/2x diluted PBS
stored slides at 4 °C under N₂-atmosphere

2015.08.29

Experiment/Protocol

spotted 3 µL of affibody (from Stockholm) and controls (GFP, bBSA)

spotting pattern:

slide-#: 500

#	spot	Concentration
1-3	200 mL culture (blue heart) Lysate	pure
4-6	200 mL culture (blue heart) Lysate	1:2 diluted
7-9	10 mL culture (red heart) Lysate	pure
10	His-GFP desalt.	~1 mg/mL
11	bBSA	0.2 mg/ml

Results

→ because of the His tag of the antigen it binds to the Ni-NTA surface→no specific antigen/affibody binding was detectable
→ retry the experiment with immobilized antigen on a PDITC surface, flush the affibody through the flow camber

Experiment 61: DNA on PDMS 6.0

protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

Considerations

prepare 3 PDMS slides for spotting with DNA and cell-free expression

2015.08.25

Experiment/Protocol

used 2 flow cells made on 18.08.2015: one with same pattern as flow cells used in iRIf (conic, one hole) and one with three holes and cavities, +1 made on 16.08.15 with Standard flow cell form and small borders
washed PDMS slides with Ethanol and H₂O
activated slides in plasma generator with 30 L/h for 1 min
incubated slides in APTES o/n at room temperature

2015.08.26

Experiment/Protocol

Flow cells were washed individually with ethanol
Washing step of 5 min in ethanol in slideholder
Flow cells were dried with wafergun
heated 90 min in oven at 70°C
cooled down with wafergun
Flow cells were incubated in PDITC for 2h
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
stored at 4°C in nitrogen atmosphere

2015.08.27

Experiment/Protocol

spots of DNA were put on flow cells according to the following pattern
incubation overnight at room temperature in humid atmosphere

spotting pattern (slide 61a):

#	sample	concentration	spot size
1	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	3 µl
2	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	5 µl
3	Cy3-HA-GFP-His-His	33 ng/µl	4 µl
4	Cy3-HA-GFP-His-His	66.1 ng/µl	4 µl

spotting pattern (slide 61b):

#	sample	concentration	spot size
1	Cy3-HA-GFP-His-His	66.1 ng/µl	5 µl
2	HA-GFP-Halo-His-His (Tobi)	99 ng/µl	3 µl
3	Cy3-HA-GFP-His-His	66.1 ng/µl	10 µl

spotting pattern (slide 61c):

#	sample	concentration	spot size
1	Cy3-HA-GFP-His-His	66.1 ng/µl	3 µl
2	Cy3-HA-GFP-His-His	33 ng/µl	4 µl
3	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	3 µl
4	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	5 µl

checked for Cy3-intensity by spotting a 1.5 µl spot of Cy3-labeled GFP (33 ng/µl) on old PDMS flow cell from 18.08.2015
spot was let dry in at room temperature until its borders were still visible
checked for fluorescence in Mircoarray Scanner

Results

Cy3 spot could be detected

2015.08.28

Measurement

slide 254: Retikulozyte, slide 012: Roth, slide 423: Koko

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer	3	60	450	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
strep-cy5	6	60	300	5 ug/ml
Buffer	7	60	300	1x

Results

Slide 12 (expressed with AG Roth mix): Platereader cy5 signal after iRIf

Slide 12 (expressed with AG Roth mix): ROI selection

Slide 12 (expressed with AG Roth mix): Binding curves

Slide 254 (expressed with retikulozyte mix): Platereader cy5 signal after iRIf

Slide 254 (expressed with retikulozyte mix): ROI selection

Slide 254 (expressed with retikulozyte mix): Binding curves

Slide 423 (expressed with Koko mix): Platereader cy5 signal after iRIf

Slide 423 (expressed with Koko mix): ROI selection

423 (expressed with Koko mix): Binding curves

Experiment 60: DNA on PDMS 5.0

protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

Considerations

prepare 2 PDMS slides for spotting with DNA

2015.08.23

Experiment/Protocol

used 2 flow cells made on 18.08.2015: one with same pattern as flow cells used in iRIf (conic, one hole) and one with three holes and cavities
washed PDMS slides with Ethanol and H₂O
activated slides in plasma generator with 30 L/h for 1 min
incubated slides in APTES o/n at room temperature

2015.08.24

each slide was washed with ethanol
a 5 min washing step in Ethanol followed
after drying with wafergun slides were baked in oven (old one) for 120 min at 70°C
slides were cooled down with wafergun
incubation in PDITC for 3h30 at RT
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
spots of DNA were put on flow cells according to the following pattern
incubation overnight at room temperature in humid atmosphere

spotting pattern (slide 60a):

#	sample	concentration	spot size
1	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	1 µl
2	Cy5-HA-GFP-His-His	12.5 ng/µl	3 µl
3	Cy5-HA-GFP-His-His	44.8 ng/µl	1 µl
4	Cy5-HA-GFP-His-His	28.7 ng/µl	0.5 µl
5	HA-GFP-Halo-His-His (Tobi)	50 ng/µl	1 µl
6	Cy5-HA-GFP-His-His	17.9 ng/µl	2 µl
7	Cy5-HA-GFP-His-His	22.4 ng/µl	3 µl
8	Cy5-HA-GFP-His-His	44.8 ng/µl	0.2 µl
9	HA-GFP-Halo-His-His (Tobi)	99 ng/µl	1 µl

spotting pattern (slide 60b):

#	sample	concentration	spot size
1	HA-GFP-Halo-His-His (Tobi)	99 ng/µl	3 µl
2	HA-GFP-Halo-His-His (Tobi)	50 ng/µl	3 µl
3	PCR-Mix von Cy5-HA-GFP-His-His (44.8 ng/µl)	-	14 µl
4	HA-GFP-Halo-His-His (Tobi)	25 ng/µl	3 µl
5	Cy5-HA-GFP-His-His	22.4 ng/µl	6 µl

2015.08.25

Experiment/Protocol

spots were let dry in at 60°C for 30 min still in humid atmosphere
expression was effected with 15 µl (60b)/7 µl (60a each flow cell) of Koko mix for 2h at 37°C
flow cells were then checked for fluorescence with microscope of AG Eimer

Results

no fluorescence could be observed

Images are upside-down, meaning the upper line is line 3 (spots 7-9)

60a

60b

2015.08.26

Results

after storage at 4°C in humid atmosphere overnight flow cells were again checked for fluorescence with microscope of AG Eimer
no fluorescence could be observed

60a

60b

Experiment 59d on slide cellfree expressed GFP on Ni-NTA slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.21

Experiment/Protocol

prepared 3 PDITC slides
Plasma activation: 40 l/h, 5 min
APTES incubation: 30 min
PDITC incubation: 3 h

2015.08.25

Experiment/Protocol

slides were sandwiched with NTA-solution and incubated o/n at 4 °C

2015.08.26

Experiment/Protocol

slides were blocked with APTES blocking solution for 1 h
slides were washed 2x with PBS
slides were incubated with NiSO₄-solution for 1 h and 20 min
slides were washed with PBS/2x diluted PBS

cell free GFP Expression mix was spotted on slides (15 µL per spot)and incubated it 3 h at 37 °C
spotted off- slide cell free expressed GFP on the two slides after the first incubation and incubated o/n at 4°C

spotting pattern:

slide-#: 303,466

#	spot	Concentration
1-3	MM + DNA
4-5	MM - DNA)
6	GFP (off-slide cell free expressed)
7	His-GFP desalt.	~1 mg/mL
8	bBSA	0.2 mg/ml

2015.08.27

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA in PBS for 5 min
blocked slides with 10 mg/mL BSA in PBS for 30 min
washed slides with PBS/diluted PBS + 20 mM imidazole/diluted PBS

Flush protocol: slide 466 and 303

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer	3	60	450	1x
Anti-GFP (goat, biotinylated)	4	30	600	3 ug/ml
Buffer	5	60	300	1x
StrepCy5	6	30	300	5 ug/ml
Buffer	7	60	300	1x

Results

ROI selection of slide 466

picture of slide 466 from the microarray scanner (exitation at 635 nm

binding curves of slide 466

→ salt deposits on the surface of slide 303 made an evaluation difficult

Experiment 59c PDITC/Ni-NTA slides for iRIf - cell free Expression on slides

protocols used :Plasma activation, iRIf slide preparation

2015.08.21

Experiment/Protocol

prepared 3 PDITC slides
Plasma activation: 40 l/h, 5 min
APTES incubation: 30 min
PDITC incubation: 3 h

2015.08.23

Experiment/Protocol

prepared 3 Ni-NTA slides according to iRIf slide preparation protocol
PDITC slides were incubated o/n with NTA at 4°C
slides were blocked in APTES blocking solution for 30 min
slides were incubated in NiSO4 for 1 h
slides were washed with PBS/diluted PBS
slides were stored at 4 °C

2015.08.25

Experiment/Protocol

spotted free expression mix on two slides (429, 424) and incubated it 3 h at 37 °C
spotting mask was accidently removed after incubation –> spotted Solutions probably mixed (no definite spots)
spotted off- slide cell free expressed GFP on the two slides after the first incubation and incubated o/n at 4°C
spotted 1 slide (500) with only off-slide cell-free expressed GFP and incubated o/n at 4°C

spotting pattern:

slide-#: 429, 424

#	spot	Concentration
1-3	MM + DNA
4-5	MM - DNA)
6	GFP (off-slide cell free expressed)
7	His-GFP desalt.	~1 mg/mL
8	bBSA	0.2 mg/ml

spotting pattern:

slide-#: 500

#	spot	Concentration
1-3	MM + DNA
4-6	MM - DNA
7-8	MM + DNA (off-slide cell free expressed, 15 µl reachtion )
10	His-GFP desalt.	~1 mg/mL
11	bBSA	0.2 mg/ml

2015.08.26

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min
slides were washed with PBS/diluted PBS + 20 mM imidazole/diluted PBS

Measurement

slide 424 and 500

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer	3	60	450	1x
Anti-GFP (goat, biotinylated)	4	30	600	3 ug/ml
Buffer	5	60	300	1x
StrepCy5	6	30	300	5 ug/ml
Buffer	7	60	300	1x

429 was not measured because the other slides showed that GFP was expressed, so the Experiment will be repeated and the above mentioned mistake (removal of spotting masks after 37 °C incubation) will be avoided.

Results

Only slide 500 was evaluated. Slide 424 and 429 showed very bad surface during measurements which was wiped off → Difficult to evaluate Slide 500: spots 4 and 8 had to be excluded from evaluation.

Slide 500: ROI selection

Slide 500: Binding curves

Experiment 59b PDITC/Ni-NTA slides for iRIf - cell free expressed Tetanus

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 2 PDITC slides (208, 216)

2015.08.21

Experiment/Protocol

prepared 2 PDITC slides
Plasma activation: 40 l/h, 5 min
APTES incubation: 30 min
PDITC incubation: 3 h

2015.08.23

Experiment/Protocol

prepared 2 Ni-NTA slides according to iRIf slide preparation protocol
PDITC slides were incubated o/n with NTA at 4°C
slides were blocked in APTES blocking solution for 30 min
slides were incubated in NiSO4 for 1 h
slides were washed with PBS/diluted PBS
slides were stored at 4 °C

2015.08.25

Experiment/Protocol

spotted with cell-free expressed Tetanus samples, GFP and bBSA

spotting pattern:

slide-#: 216

#	spot	Concentration
1	B+ (1)
2	B+ (2)
3	B+ (3)
4	B- (1)
5	B- (2)
6	K+ (1)
7	His-GFP desalt.	~0.5 mg/mL
8	His-GFP lysate
9	bBSA	0.2 mg/ml
10	K-
11	BSA

slide-#: 208

#	spot	Concentration
1	K+ (1)
2	K+ (2)
3	K+ (3)
4	K- (1)
5	K- (2)
6	B+ (1)
7	His-GFP desalt.	~0.5 mg/mL
8	His-GFP lysate
9	bBSA	0.2 mg/ml
10	B-
11	BSA

2015.08.26

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min
slides were washed with PBS/diluted PBS + 20 mM imidazole/diluted PBS

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer (PBS)	3	60	450	1x
Anti-Tetanus (goat, pk)	4	30	600	25 ug/ml
Buffer (PBS)	5	60	300	1x
Anti-GFP (biotinylated, Goat)	6	30	600	3 ug/ml
Buffer (PBS)	7	60	300	1x
Anti-Goat	8	30	600	5 ug/ml
Buffer (PBS)	9	60	300	1x
StrepCy5	10	30	300	5 ug/ml
Buffer (PBS)	11	60	300	1x

Results

Slide 208: Only 5 spots (spot 3, 5, 7, 8 and 9) could be evaluated.

Slide 208: ROI selection

Slide 216: ROI selection

Slide 208: Binding curves

Slide 216: Binding curves

Experiment 59a: PDITC/Ni-NTA slides for iRIf - Tetanus with own blood

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 2 PDITC slides

2015.08.21

Experiment/Protocol

prepared 2 PDITC slides
Plasma activation: 40 l/h, 5 min
APTES incubation: 30 min
PDITC incubation: 3 h
spotted 3 µL of Antigen and controls (GFP)

spotting pattern:

slide-#: 287, 504

#	spot	Concentration
1-2	Tetanus (11) desalt	460 µg/mL
3-4	Tetanus (11) desalt	100 µg/mL
5-6	Tetanus (11) desalt	20 µg/mL
7-8	His-GFP desalt.	~0.5 mg/mL
9	bBSA	0.2 mg/ml
10-11	Tetanus (11) lysate

2015.08.22

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
slides were blocked with 10 mg/mL BSA in PBS for 30 min
slides were washed with Aqua dest and dried

Measurement

Flush protocol slide 287

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	900	10 mg/ml
Buffer (PBS)	3	60	600	1x
Serum rabbit Anti-GFP	4	30	600	1:330
Buffer (PBS)	5	60	300	1x
Serum (+) SIG002	6	30	600	1:10
Buffer (PBS)	7	60	300	1x
Anti-Human	8	60	300	1 ug/ml
Buffer (PBS)	9	60	300	1x

no Strep-Cy5 was flushed over slide –> no scanner Pictures

Flush protocol slide 504

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	900	10 mg/ml
Buffer (PBS)	3	60	600	1x
Serum (-) SIG002	4	30	600	1:10
Buffer (PBS)	5	60	300	1x
Serum (+) SIG002	6	30	600	1:10
Buffer (PBS)	7	60	300	1x
Anti-Human	8	60	300	1 ug/ml
Buffer (PBS)	9	60	300	1x
Anti-GFP (biotinylated, Goat)	10	60	300	1 ug/ml
Buffer (PBS)	11	60	300	1x
Strep-Cy5	12	60	300	5 ug/ml
Buffer (PBS)	13	60	300	1x

Results

Slide 287: Selection of ROI

Slide 287: Binding curves

Slide 504: Selection of ROI

Slide 504: Binding curves

→ detection of Tetanus antibodys from blood serum after vaccination is possible
→ huge cross reactivity with bBSA spot→probably due to biodinidase enzyme

Experiment 58: ELISA with Tetanus-Toxin and blood

Considerations

Check if antibodies in blood of freshly vaccinated Patient SIG002 binds to our self-expressed Tetanus Antigen
detection of antibodies in blood with anti-human (mouse)
detection of anti-human with HRP-labeled anti-mouse

2015.08.20

Experiment/Protocol

100 µL of each Tet-Tox and bBSA concentration were pipetted into maxisorb-plate

	1	2	3
A	Tet-Tox 100 µg/mL	bBSA 100 µg/mL	bBSA 0.4 µg/mL
B	Tet-Tox 50 µg/mL	bBSA 50 µg/mL	bBSA 0.2 µg/mL
C	Tet-Tox 25 µg/mL	bBSA 25 µg/mL	bBSA 0.1 µg/mL
D	Tet-Tox 12 µg/mL	bBSA 12 µg/mL	bBSA 0.05 µg/mL
E	Tet-Tox 6 µg/mL	bBSA 6 µg/mL	bBSA 0.025 µg/mL
F	Tet-Tox 3 µg/mL	bBSA 3 µg/mL	bBSA 0.012 µg/mL
G	Tet-Tox 1.5 µg/mL	bBSA 1.5 µg/mL	bBSA 0.006 µg/mL
H	neg (PBS)	bBSA 0.8 µg/mL	neg (PBS)

2015.08.21

Experiment/Protocol

8:34 am: all wells exept H1 were blocked with 5 mg/mL BSA in PBS
wells were washed with PBS 2 times for ~2 min
10:00 am: 100 µL of 1/10 diluted blood Serum were added to well A1-H1 and again 100 µL of 5 mg/mL BSA in PBS to A2-H2 and A3-H3
all solutions were overlayed with 100 µL PBS
wells were washed with PBS 3 times for ~2 min
11.07 am: 100 µL of anti-human (1 µg/mL) were added to well A1-H1 and again 100 µL of 5 mg/mL BSA in PBS to A2-H2 and A3-H3
all solutions were overlayed with 100 µL PBS
wells were washed with PBS 3 times for ~2 min
1:00 pm: 100 µL of HRP-anti-mouse (1 µg/mL) were added to well A1-H1 and 100 µL of HRP-Strep (0.5 µg/mL) to A2-H2 and A3-H3
all solutions were overlayed with 100 µL PBS
wells were washed with PBS 5 times for ~2 min

Hydrogenperoxide and 3,3´,5,5´-Tetramethylbenzidine were added to each well and the Absorption was measured in the plate reader

Results

Absorbance over time for Tetanus-ELISA Test with own blood

Experiment 57d: Ni NTA iRIf slides - Salmonella

protocols used :Plasma activation, iRIf slide preparation

2015.08.18

Considerations

prepare 2 PDITC iRif slides
slide#:

Experiment/Protocol

slides were washed according to iRIf slide wash steps
Plasma activation: 40 L/h 5 min
APTES incubation: 30 min
PDITC incubation 3.5 h

2015.08.20

slides were blocked in APTES blocking solution for 45 min
slides were incubated in NiSO₄ for 1 h
slides were washed with PBS/diluted PBS
slides were stored at 4 °C

2015.08.21

* spotted 3 µL of Salmonella desalt and lysate and controls (GFP-Lysate, GFP-desalt, bBSA)

spotting pattern:

slide-#: 012, 426

#	spot	Concentration
1-3	Salmonella (15) desalt
4-6	Salmonella (15) lysate
7	His-GFP desalt.	0.2 mg/ml
8	His-GFP Lysate	~ 0.5 mg/ml
9	His-GFP Lysate	~ 0.5 mg/ml
10	nt-GFP Lysate	~ 0.5 mg/ml
11	bBSA	0.2 mg/ml

2015.08.22

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in TBS for 5 min
slides were blocked with 10 mg/mL BSA in TBS for 30 min
slides were washed with TBS/diluted TBS + 20 mM imidazole/diluted TBS

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS)	1	60	720	1x
His-HIV-Lysate (17)/BSA	2	60	900	1:1
Buffer (TBS)	3	60	600	1x
Anti-GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (TBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (TBS)	7	60	300	1x
Anti-Salmonella desalt (13)	8	30	600	~60 µg/mL
Buffer (TBS)	9	60	300	1x
Anti-Salmonella Lysate (13)	10	30	600	~60 µg/mL
Buffer (TBS)	11	60	300	1x

Experiment 57c: Ni NTA iRIf slides - cell free expressed GFP

protocols used :Plasma activation, iRIf slide preparation

2015.08.18

Considerations

prepare 4 PDITC iRif slides
slide#: 423, 215, 254, 024

Experiment/Protocol

slides were washed according to iRIf slide wash steps
Plasma activation: 40 L/h 5 min
APTES incubation: 30 min
PDITC incubation 3.5 h

2015.08.20

slides were blocked in APTES blocking solution for 45 min
slides were incubated in NiSO₄ for 1 h
slides were washed with PBS/diluted PBS
spotted 3 µL of cell free expressed GFPs and controls (GFP-Lysate, GFP-desalt, bBSA)

spotting pattern:

slide-#: 423, 254

#	spot	Concentration
1	BK HA-GFP-His6His6 (E2)
2	KK HA-GFP-His6His6 (G2)
3	PP HA-GFP-His6His6 (I2)
4	BP HA-GFP-His6His6 (K2)
5	BK His10-GFP-Spy (E3)
6	KK His10-GFP-Spy (G3)
7	His-GFP desalt.	0.2 mg/ml
8	His-GFP Lysate	~ 0.5 mg/ml
9	bBSA	0.2 mg/ml
10	PP His10-GFP-Spy (I3)
11	BP His10-GFP-Spy (K3)

slide-#: 024, 215

#	spot	Concentration
1	BK HA-GFP-His6His6 (F2)
2	KK HA-GFP-His6His6 (H2)
3	PP HA-GFP-His6His6 (J2)
4	BP HA-GFP-His6His6 (L2)
5	BK His10-GFP-Spy (F3)
6	KK His10-GFP-Spy (H3)
7	His-GFP desalt.	0.2 mg/ml
8	His-GFP Lysate	~ 0.5 mg/ml
9	bBSA	0.2 mg/ml
10	PP His10-GFP-Spy (J3)
11	BP His10-GFP-Spy (L3)

2015.08.21

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min
slides were washed with PBS/diluted PBS + 20 mM Imidazole/diluted PBS

Measurement

slide 215: old Setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

slide_024_binding_curve

slide_024_roi_024

slide_215_binding_curves

slide_215_roi

slide_254_binding_curves

slide_254_roi

slide_423_binding_curves

slide_423_roi

Experiment 57b: Ni NTA iRIf slides - cell free Expression on Ni-NTA slides

protocols used :Plasma activation, iRIf slide preparation

2015.08.18

Considerations

prepare 2 PDITC iRif slides
slide#: 466, 253

Experiment/Protocol

slides were washed according to iRIf slide wash steps
Plasma activation: 40 L/h 5 min
APTES incubation: 30 min
PDITC incubation 3.5 h

2015.08.20

slides were blocked in APTES blocking solution for 45 min
slides were incubated in NiSO₄ for 1 h
slides were washed with PBS/diluted PBS
two slides (466, 253) were spotted with 15 µl per spot spottingmasks

Spotting:

#	spot	Concentration
1-3	His-GFP (cellfree expressed))
4-6	neg. control (cellfree Expression without DNA)
7	bBSA	200 µg/ml
8	His GFP lysate

slides were covered with glass slides to avoid evaporation
slides were incubated for for 3 h at 37 °C
measurement in iRIf

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

Slide 253: Quotion picture

Slide 466: Quotion picture

→ no cell free expressed GFP could be detected
→ on slide 253 even the spotted GFP lysate was not detectable, this is probably due to repeaded problems with the correct flooding of the flow cell during the measurement with the iRIf setup
→ the experiment will be repeated with additional spots of „off slide“ cell free expressed GFP

Experiment 57a: PDITC iRIf slides - anti-GFP-Serum measurement

2015.08.18

Considerations

prepare 2 PDITC iRif slides
slide#: 315, 502

Experiment/Protocol

slides were washed according to iRIf slide wash steps
Plasma activation: 40 L/h 5 min
APTES incubation: 30 min
PDITC incubation 3.5 h
slides were washed and dried with wafer gun
3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

#	spot	Concentration
1-2	His-GFP (cellfree expressed))
3	His-GFP desalt.	0.2 mg/ml
4-5	neg. control (cellfree Expression without DNA)
6-7	His-mCherry lysate	1:10
8-9	His-GFP lysate	1:10
10	BSA	0.2 mg/ml
11	bBSA	0.2 mg/ml

slides were measured with Serum(-) and Serum(+)

2015.08.19

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA for 5 min
blocked slides with 10 mg/mL BSA for 30 min
washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Measurement

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
Serum (-)	4	30	600	*
Buffer	5	60	300	1x
Serum (+)/a-GFP	6	30	600	* / 3ug/ml
Buffer	7	60	600	1x
anti-Rabbit	8	30	600	5 ug/ml
Buffer	9	60	600	1x
Strep Cy5	10	30	6000	5 ug/ml
Buffer	11	60	600	1x

Results

Serum (-) / Serum (+)

Slide 315: Selection of ROIs

Slide 315: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

Experiment 56: DNA on PDMS 4.0

protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

Considerations

prepare 2 PDMS slides for spotting with DNA

2015.08.17

Experiment/Protocol

used 2 flow cells made on 16.08.2015 with same pattern as flow cells used in iRIf (conic, one hole, one with pillars/one bad)
washed PDMS slides with Ethanol and H₂O
activated slides in plasma generator with 30 L/h for 1 min
incubated slides in APTES o/n at room temperature

2015.08.18

each slide was washed with ethanol
a 5 min washing step in Ethanol followed
after drying with wafergun slides were baked in oven (better one) for 90 min at 70°C
slides were cooled down with wafergun
incubation in PDITC for 2h at RT
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
flow cells were spotted with 1 µl spots and incubated over night at room temperature in humid atmosphere

spotting pattern:

#	spot	concentration
1	Cy5-HA-GFP-His-His	12.5 ng/µl
2	Cy5-HA-GFP-His-His	17.9 ng/µl
3	His-tYFP-Spy (lina104)	25 ng/µl
4	HA-GFP-Halo-His-His (Tobi)	25 ng/µl
5	Cy5-HA-GFP-His-His	22.4 ng/µl
6	PCR-Mix	-

2015.08.19

Experiment/Protocol

expression was effected with 15 µl of Koko mix for 2h at 37°C
flow cells were then checked for fluorescence with microscope of AG Eimer

Results

for both flow cells no fluorescence could be observed

56a

56b

Experiment/Protocol

flow cells were disassembled and washed with PBS and H₂O
dried with wafergun
expression was repeated with 15 µl of EasyExpress-Mix (AG Roth) at 37°C in humid atmosphere
fluorescence was measured after 1h and 2h with microscope of AG Eimer

Results

a few fluorescent spots could be observed

56a after 1h

56a after 2h

56b after 1h

56b after 2h

Experiment 55: DNA on PDMS 3.0

protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

Considerations

prepare 2 PDMS slides for spotting with DNA

2015.08.15

Experiment/Protocol

used 2 flow cells (one with same pattern as flow cells used in iRIf and pillars, one with cavities and three lines)
washed PDMS slides with Ethanol and H₂O
used correct APTES solution (1 mL APTES, 1 mL H₂O in 18 mL Ethanol)
activated slides in Plasma Generator with 40 L/h for 1 min
incubated slides in APTES o/n

2015.08.16

each slide was washed with ethanol
a 5 min washing step in Ethanol followed
after drying with wafergun slides were baked in oven (better one) for 90 min at 70°C
slides were cooled down with wafergun
incubation in PDITC for 3h at RT
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
cells were spotted with 3 µl spots and incubated over night at room temperature in humid atmosphere
sample 1 was diluted 1:2 (original oncentration 44,8 ng/µl) in NaPi buffer 150 mM

spotting pattern:

flow cell 55a

flow cell 55b

#	spot	concentration
1	Cy5-HA-GFP-His-His	22,4 ng/µl
2	Control:HA-GFP-Halo-His-His	25 ng/µl
3	PCR-Mix of 1	-

2015.08.17

Petri dish with flow cells was put in oven at 60°C for 30 min
Flow cells were then washed with aqua dest. and dried with wafergun
Expression was effected for 20, 40 and 130 min with AG Roth expression mix at 37°C (not sealed, no humid atmosphere)
flow cells were checked for fluorescence in microscope of AG Eimer
afterwards flow cells were washed with 2 ml PBS and aqua dest. and thene dried with wafergun
stored in petri dish sealed with parafilm at 4°C

Results

Expression of our GFP conctruct and the control construct could be observed on both flow cells
Expressed GFP seems to diffuse with the time. This might be due to the transport of the slides from the incubator to the microscope and back.

55a after 20 min incubation time

55a after 40 min incubation time

55a after 130 min incubation time

55b after 20 min incubation time

2015.08.18

Expression was effected for 2h with Koko expression mix at 37°C (sealed, humid atmosphere)
flow cells were checked for fluorescence in microscope of AG Eimer
afterwards flow cells were washed with 2 ml PBS and aqua dest. and thene dried with wafergun
stored in petri dish sealed with parafilm at 4°C

Results

no fluorescence could be observed for both flow cells as well as for Tobis 4 flow cells

55a after 2h

55b after 2h

Experiment 54b: Ni-NTA iRIf slides - rabbit Serum

protocols used :Plasma activation, iRIf slide preparation

2015.08.15

Considerations

prepare 4 PDITC iRIf slides
slides : 504, 500, 216, 208

Experiment/Protocol

4 PDITC iRIf slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h

2015.08.17

Experiment/Protocol

slides were sandwiched with 60 µL NTA-solution and incubated o/n at 4 °C

2015.08.18

Experiment/Protocol

slides were blocked in APTES blocking solution for 1 h
slides were incubated in NiSO<Sub>4</Sub> for 1 h
slides were washed with PBS/diluted PBS
3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

#	spot	Concentration
1-2	His-GFP (cellfree expressed))
3	His-GFP desalt.	0.2 mg/ml
4-5	neg. control (cellfree Expression without DNA)
6-7	His-mCherry lysate	1:10
8-9	His-GFP lysate	1:10
10	BSA	0.2 mg/ml
11	bBSA	0.2 mg/ml

3 of the slides (208, 216, 504) were measured with Serum(-) and Serum(+)
1 of the slides (500) was measured with Serum(-) and anti-GFP
(slide from Exp. 50c was spotted similar and also measured with Serum(-) and anti-GFP)

2015.08.19

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA for 5 min
blocked slides with 10 mg/mL BSA for 30 min
washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Measurement

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
Serum (-)	4	30	600	*
Buffer	5	60	300	1x
Serum (+)/a-GFP	6	30	600	* / 3ug/ml
Buffer	7	60	600	1x
anti-Rabbit	8	30	600	5 ug/ml
Buffer	9	60	600	1x
Strep Cy5	10	30	6000	5 ug/ml
Buffer	11	60	600	1x

Results

Serum (-) / Serum (+)

Slide 208: Selection of ROIs

Slide 208: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

Serum (-) / a-GFP

Slide 424: Selection of ROIs

Slide 424: Binding curves

The relative intensity shift for the three approaches (Serum(-)Serum(+) on Ni-NTA ; Serum(-)Serum(+) on PDITC; Serum(-)a-GFP on Ni-NTA) were compared. As expected and seen in the binding curves cellfree expression Mix (His-GFP) shows no signal on PDITC (other proteins within the mix bind to the surface) but on specific Ni-NTA surface. Taken amount of serum performs less (2-3 fold) than 3 ug/ml a-GFP.

Evaluation of the relative intensity shift. From left to right spot 1-11. For each spot rel. intensity shift is plotted with bars for Serum(-)flush and Serum(+)/a-GFP

Emission within 532nm channel (mCherry) after iRIf measurement. Averaged over all samples.

Emission within 635nm channel (Cy5) after iRIf measurement. Averaged over all samples

Experiment 54a: PDITC iRIf slides - own device

protocols used :Plasma activation, iRIf slide preparation

2015.08.15

Considerations

prepare 1 PDITC iRIf slides
slides : 467

Experiment/Protocol

1 PDITC iRIf slides were prepared
Plasma activation: 40 L/h, 5 min
APTES incubation: 30 min
PDITC incubation: 2 h

2015.08.17

Experiment/Protocol

1 slide was spotted alternating with bBSA and BSA for measurement in own device, in total 14 spots

2015.08.18

Experiment/Protocol

slide for own device was blocked (2x 10 mg/mL BSA on spots for 5 min, 1x in 10 mg/mL BSA for 30 min)

Results

Experiment 53b: Plasma activated slides with PLL-PEG-HTL from Piehler

protocols used :Plasma activation, Halosurface from AG Piehler

2015.08.14

Considerations

second try with halo surface

Experiment/Protocol

activated slides 253 and 254 with 40 L/h for 5 min
cross-sandwiched slides with 15 µL PLL-PEG-HTL
incubated slides for 10 min
washed slides with Aqua dest and stored them in slideholder o/n (no N₂-atmosphere)

2015.08.16

Experiment/Protocol

blocked slides in 10 mg/ml BSA for 1h
washed slides with aqua dest. and dried with nitrogengun
spotted slides and incubated for 1 h at roomtemperature

Spotting:

Spot	Protein	Concentration
1-2	Halo GFP	0.5 mg/ml
3-4	Halo GFP	0.1 mg/ml
5-6	Halo GFP	0.02 mg/ml
7	bBSA	0.2 mg/ml
8	Halo mCherry	0.5 mg/ml
9	His-GFP (Max)	0.5 mg/ml
10	His Halo GFP (Piehler, pos. control)	0.25 mg/ml
11	His GFP (Piehler, neg. control)	0.25 mg/ml

blocked wells twice with 10 mg/ml BSA for 5 min
blocked slides 30 min in 10 mg/ml BSA
washed slides with water and dried with nitrogengun
measured fluorescence intensity with fluorescence microscope
measurement in iRIf

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

Slide 254: Quotion picture

Slide 254: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 253: Quotion picture

Slide 253: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Experiment 53a: Plasma activated slides with abcr-silane

protocols used :Plasma activation, Halosurface from ABCR

2015.08.14

Considerations

second try with halo surface

Experiment/Protocol

activated slides 012 and 215 with 40 L/h for 5 min
sandwiched slides with 80 µL abcr-silane
incubated slides for 1 h
washed slides with Aqua dest and stored them in slideholder o/n (no N₂-atmosphere)

2015.08.16

Experiment/Protocol

blocked slides in 10 mg/ml BSA for 1h
washed slides with aqua dest. and dried with nitrogengun
spotted slides and incubated for 1 h at roomtemperature

Spotting:

Spot	Protein	Concentration
1-2	Halo GFP	0.5 mg/ml
3-4	Halo GFP	0.1 mg/ml
5-6	Halo GFP	0.02 mg/ml
7	bBSA	0.2 mg/ml
8	Halo mCherry	0.5 mg/ml
9	His-GFP (Max)	0.5 mg/ml
10	His Halo GFP (Piehler, pos. control)	0.25 mg/ml
11	His GFP (Piehler, neg. control)	0.25 mg/ml

blocked wells twice with 10 mg/ml BSA for 5 min
blocked slides 30 min in 10 mg/ml BSA
washed slides with water and dried with nitrogengun
measured fluorescence intensity with fluorescence microscope
measurement in iRIf

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

Slide 012: Quotion picture

Slide 012: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 215: Quotion picture

Slide 215: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Experiment 52c: PDITC Slides

protocols used :Plasma activation, iRIf slide preparation

2015.08.14

Considerations

Experiment/Protocol

washed 1 iRIf slides
Plasma activation: 40 L/h, 5 min
APTES incubation: 45 min
PDITC incubation: 2 h
stored slides at 4 °C o/n

2015.08.17

Experiment/Protocol

slide was spotted alternating with bBSA and BSA for measurement in own device, in total 14 spots

2015.08.18

Experiment/Protocol

slide was blocked (2x 10 mg/mL BSA on spots for 5 min, 1x in 10 mg/mL BSA for 30 min)

Results

Experiment 52b: PDITC Slides with Wiesmüller Halo-Linker 2

protocols used :Plasma activation, iRIf slide preparation, Halosurface from Wiesmüller

2015.08.14

Considerations

Experiment/Protocol

washed 2 iRIf slides
Plasma activation: 40 L/h, 5 min
APTES incubation: 45 min
PDITC incubation: 2 h
stored slides at 4 °C o/n

slide 423 and 287 were incubated with Wiesmüller Halo-Linker 2 o/n in slideholder

2015.08.16

Experiment/Protocol

washed slides with Aqua dest. and dried with nitrogengun
blocked slides in 10 mg/ml BSA for 1h
washed slides with aqua dest. and dried with nitrogengun
spotted slides and incubated for 1 h at roomtemperature

Spotting:

Spot	Protein	Concentration
1-2	Halo GFP	0.5 mg/ml
3-4	Halo GFP	0.1 mg/ml
5-6	Halo GFP	0.02 mg/ml
7	bBSA	0.2 mg/ml
8	Halo mCherry	0.5 mg/ml
9	His-GFP (Max)	0.5 mg/ml
10	His Halo GFP (Piehler, pos. control)	0.25 mg/ml
11	His GFP (Piehler, neg. control)	0.25 mg/ml

blocked wells twice with 10 mg/ml BSA for 5 min
blocked slides 30 min in 10 mg/ml BSA
washed slides with water and dried with nitrogengun
measured fluorescence intensity with fluorescence microscope
measurement in iRIf

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

Slide 423: Quotion picture

Slide 423: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 287: Quotion picture

Slide 287: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Experiment 52a: PDITC Slides with Wiesmüller Halo-Linker 1

protocols used :Plasma activation, iRIf slide preparation, Halosurface from Wiesmüller

2015.08.14

Considerations

Experiment/Protocol

washed 2 iRIf slides
Plasma activation: 40 L/h, 5 min
APTES incubation: 45 min
PDITC incubation: 2 h
stored slides at 4 °C o/n

slide 426 and 466 were incubated with Wiesmüller Halo-Linker 1 o/n in slideholder

2015.08.16

Experiment/Protocol

washed slides with Aqua dest. and dried with nitrogengun
blocked slides in 10 mg/ml BSA for 1h
washed slides with aqua dest. and dried with nitrogengun
spotted slides and incubated for 1 h at roomtemperature

Spotting:

Spot	Protein	Concentration
1-2	Halo GFP	0.5 mg/ml
3-4	Halo GFP	0.1 mg/ml
5-6	Halo GFP	0.02 mg/ml
7	bBSA	0.2 mg/ml
8	Halo mCherry	0.5 mg/ml
9	His-GFP (Max)	0.5 mg/ml
10	His Halo GFP (Piehler, pos. control)	0.25 mg/ml
11	His GFP (Piehler, neg. control)	0.25 mg/ml

blocked wells twice with 10 mg/ml BSA for 5 min
blocked slides 30 min in 10 mg/ml BSA
washed slides with water and dried with nitrogengun
measured fluorescence intensity with fluorescence microscope
measurement in iRIf

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer (PBS)	3	60	600	1x
Anti GFP (biotinylated, goat)	4	30	600	5 ug/ml
Buffer (PBS)	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer (PBS)	7	60	300	1x

Results

Slide 426: Selection of ROI

Slide 426: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 426: Binding curves

Slide 466: Selection of ROI

Slide 466: Picture obtained from fluorescence scanner with enhanced brightness. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 466: Binding curves

Experiment 47b/49c: PDITC for iRIF/new Device

protocols used :Plasma activation, iRIf slide preparation

2015.08.13

Considerations

test if antibody binding is detectable with self built iRIf setup

Experiment/Protocol

used two already prepared iRIf PDITC slides from Exp. 47b (265) and 49c (503)
spotted bBSA (200 µg/ml) and BSA (10 mg/ml) in an alternating pattern on slide 503
spotted anti HCV antibodys (rabbit, polyclonal, 500 µg/ml)and BSA (10 mg/ml) an alternating pattern on slide 265
spotted 18 spots in total on each slide

Results

Experiment 51: DNA on PDMS 2.0

protocols used : PDMS-PDITC

2015.08.12

Considerations

repeat immobilization of Cy5-labeled DNA on PDMS slides with correct APTES solution

Experiment/Protocol

used 2 flow chamber slides with same pattern as flow Chambers used in iRIf (conic, one hole)
washed PDMS slides with Ethanol and H₂O
used correct APTES solution (1 mL APTES, 1 mL H₂O in 18 mL Ethanol)
activated slides in Plasma Generator with 40 L/h for 1 min
incubated slides in APTES o/n

2015.08.13

each slide was washed with ethanol
a 5 min washing step in Ethanol followed
after drying with wafergun slides were baked in oven for 90 min at 70°C
slides were cooled down with wafergun
incubation in PDITC for 5h40min at RT
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
1 slide (51a) was spotted with 1 µl spots and incubated over night
sample 1 was diluted 1:4,64 (original oncentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 51a:

#	spot	concentration
1	His-tYFP-Spy (lina104) (top 0.5 µl, bottom spot 1 µl)	25 ng/µl
2	His-tYFP-Halo (lina105)	20 ng/µl
3	HA-GFP-His₆-His₆	28.7 ng/µl
4	Control by Tobi: HA-GFP-Halo-His	25 ng/µl

slide (b) was spotted with 3 µl spots and incubated for 2 nights at room temperature
sample 1 was diluted 1:4,64 (original concentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 51b:

#	spot	concentration
1	His-tYFP-Spy (lina104)	25 ng/µl
2	His-tYFP-Halo (lina105)	20 ng/µl
3	HA-GFP-His₆-His₆	28.7 ng/µl
4	Control by Tobi: HA-GFP-Halo-HisHis	25 ng/µl

2015.08.14

Experiment/Protocol

DNA was let dry in
slide 51a was washed with H₂O and dried with wafergun

Results

slide 51a was measured in Microarray Scanner for Cy5
no spots could be seen

2015.08.15

Experiment/Protocol

DNA was let dry in
slide 51b was washed with H₂O and dried with wafergun

Results

measured in Microarray Scanner for Cy5
3 spots could be (hardly) seen with the eye. The intensity was too low to be visible on the derived image file. Background was approximately at 0.7 to 1.7. The spot was between 1.2 and 4.0.

2015.08.17

Experiment/Protocol

15 µl of cell-free expression mix (EasyExpress (Kubick) of AG Roth) were added to each flow cell
incubation for 20 min at 37°C
fluorescence was measured in microscope of AG Eimer
no expressed GFP could be detected

Experiment 50c: Ni-NTA slides for iRIf - rabbit serum

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 1 Ni-NTA slides for measurement in iRIf
slide-#: 424

Experiment/Protocol

washed 1 iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
stored slides at 4 °C o/n

2015.08.12

Experiment/Protocol

sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

2015.08.13

Experiment/Protocol

blocked slides in APTES/PDITC blocking solution for 1 h
washed 2x with PBS
incubated slides for 1h 15 min in NiSO<Sub>4</Sub>-solution
washed slides 2x with PBS
washed slides with 1/10 diluted PBS
stored slides for later use under N₂-atmosphere at 4 °C

2015.08.18

Experiment/Protocol

3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

#	spot	Concentration
1-2	His-GFP (cellfree expressed))
3	His-GFP desalt.	0.2 mg/ml
4-5	neg. control (cellfree Expression without DNA)
6-7	His-mCherry lysate	1:10
8-9	His-GFP lysate	1:10
10	BSA	0.2 mg/ml
11	bBSA	0.2 mg/ml

slide (424) was measured with Serum(-) and anti-GFP (measured together with slides from Exp. 54b)

2015.08.19

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA for 5 min
blocked slides with 10 mg/mL BSA for 30 min
washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Measurement

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
Serum (-)	4	30	600	*
Buffer	5	60	300	1x
Serum (+)/a-GFP	6	30	600	* / 3ug/ml
Buffer	7	60	600	1x
anti-Rabbit	8	30	600	5 ug/ml
Buffer	9	60	600	1x
Strep Cy5	10	30	6000	5 ug/ml
Buffer	11	60	600	1x

Results

Experiment 50b: Ni-NTA slides for iRIf - Antigens and GFP lysates + purified (more HCV)

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 2 Ni-NTA slides for measurement in iRIf
Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
also use TBS for iRIf measurement

Experiment/Protocol

washed 2 iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
stored slides at 4 °C o/n

2015.08.12

Experiment/Protocol

sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

2015.08.13

Experiment/Protocol

blocked slides in APTES/PDITC blocking solution for 1 h
washed 2x with PBS
incubated slides for 1h 15 min in NiSO₄ -solution
washed slides 2x with PBS
washed slides with 1/10 diluted PBS
spotted antigens were diluted in TBS
spotted slides 500 and 501

Spotting:

Spot	Protein	Number of Spots	Concentration
1-2	pET_1003 (HCV) lysate	2	~0.08 mg/ml
3-4	pET_1003 (HCV) desalt	2	~0.33 mg/ml
5	pET_1703 (HIV) lysate	1	~0.15 mg/ml
6	pET_1703 (HIV) desalt	1	~0.62 mg/ml
7	His-GFP (Max)	1	0.5 mg/ml
8	His-GFP lysate	1	~0.2 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	His-GFP aufgereinigt	2	~0.5 mg/ml

2015.08.14

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in TBS for 5 min
slides were blocked with 10 mg/mL BSA in TBS for 30 min
slides were washed with TBS
slides were washed with 1/10 diluted TBS + 20 mM imidazole
slides were washed with 1/10 diluted TBS

Measurement

slide 500 was measured in new setup
slide 501 could not be measured, air bubbles appeared every time the slide was flushed –> surface must be really hydrophobic. Maybe slide was destroyed during tests with PDMS-stamps.

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	600	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-HIV (monoclonal,mouse)	10	30	600	10 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-HCV (monoclonal,mouse)	12	30	600	10 ug/ml
Buffer (TBS!!!)	13	60	600	1x
Anti-His	14	30	600	5 ug/ml
Buffer (TBS!!!)	15	60	600	1x
Anti-GFP (biotinylated, goat)	16	30	600	3 ug/ml
Buffer (TBS!!!)	17	60	600	1x
StrepCy5	18	30	600	5 ug/ml
Buffer (TBS!!!)	19	60	600	1x

Results

Experiment 50a: Ni-NTA slides for iRIf - Antigens and GFP lysates + purified (more HIV)

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 2 Ni-NTA slides for measurement in iRIf
Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
also use TBS for iRIf measurement

Experiment/Protocol

washed 2 iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
stored slides at 4 °C o/n

2015.08.12

Experiment/Protocol

sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

2015.08.13

Experiment/Protocol

blocked slides in APTES/PDITC blocking solution for 1 h
washed 2x with PBS
incubated slides for 1h 15 min in NiSO₄-solution
washed slides 2x with PBS
washed slides with 1/10 diluted PBS
antigenes were diluted in TBS
spotted slide 216 and 467

Spotting:

Spot	Protein	Number of Spots	Concentration
1-2	pET_1703 (HIV) lysate	2	~0.15 mg/ml
3-4	pET_1703 (HIV) desalt	2	~0.62 mg/ml
5	pET_1003 (HCV) lysate	1	~0.08 mg/ml
6	pET_1003 (HCV) desalt	1	~0.33 mg/ml
7	His-GFP (Max)	1	0.5 mg/ml
8	His-GFP desalt	1	~0.5 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	His-GFP lysate	2	~0.2 mg/ml

2015.08.14

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
slides were blocked with 10 mg/mL BSA in TBS for 30 min
slides were washed with TBS
slides were washed with 1/10 diluted TBS + 20 mM imidazole
slides were washed with 1/10 diluted TBS

Measurement

216 was measured in old Setup´
467 was measured in new Setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	600	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-HIV (monoclonal,mouse)	10	30	600	10 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-HCV (monoclonal,mouse)	12	30	600	10 ug/ml
Buffer (TBS!!!)	13	60	600	1x
Anti-Mouse	14	30	600	5 ug/ml
Buffer (TBS!!!)	15	60	600	1x
Anti-GFP (biotinylated, goat)	16	30	600	3 ug/ml
Buffer (TBS!!!)	17	60	600	1x
StrepCy5	18	30	600	5 ug/ml
Buffer (TBS!!!)	19	60	600	1x

Results

Slide 24: Selection of ROIs: 1 HIV spot (spot 2) and one HCV spot (spot 3) were excluded due to high amounts of salt on it

Slide 24: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves for all spots

Slide 502: Binding curve (second Salmonella spot excluded)

Results: We have specific antibody/antigen binding for Salmonella! (a-HIV/HCV do not bind…) Slide 502: During measurement within the first buffer step/blocking step most of the second Salmonella spot was washed away. Related to this the spot behaves strange. Slide 24: Also anti-salmonella lysate was tested. Here we see (as expected) stronger unspecific binding at other spots (nevertheless greatest binding for the salmonella spots). The unspecific/transient binding proteins get mostly washed away during the following buffer step.

Slide 24: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

Slide 502: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

Experiment 49c: PDITC slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 1 PDITC slides for measurement in iRIf

Experiment/Protocol

washed 1 iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
stored slides at 4 °C under N₂-atmosphere

2015.08.13

used slides for measurement in own device –> Exp. 52

Experiment 49b: PDITC slides for iRIf - Antigens

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 2 PDITC slides for measurement in iRIf
Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
also use TBS for iRIf measurement

Experiment/Protocol

washed 2 iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
stored slides at 4 °C under N₂-atmosphere

2015.08.13

Experiment/Protocol

spotted slides 502 and 024
incubated at 4°C o/n

Spotting:

Spot	Protein	Number of spots	Concentration
1-2	pET_1003 (HCV) desalt	2	~0.33 mg/ml
3-4	pET_1703 (HIV) desalt	2	~0.62 mg/ml
5-6	pIG_1501 (Salmonella) desalt	2	~0.62mg/ml
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His desalt	1	0.5 mg/ml
9	GFP-His lysate	1	~0.2 mg/ml
10	bBSA	1	0.2 mg/ml
11	BSA	1	0.2 mg/ml

2015.08.13

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS (should have used TBS) for 5 min
slides were blocked with 10 mg/mL BSA in PBS for 30 min

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	300	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-HIV (monoclonal,mouse)	10	30	600	10 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-HCV (monoclonal,mouse)	12	30	600	10 ug/ml
Buffer (TBS!!!)	13	60	600	1x
Anti-Mouse	14	30	600	5 ug/ml
Buffer (TBS!!!)	15	60	600	1x
Anti-GFP (biotinylated, goat)	16	30	600	3 ug/ml
Buffer (TBS!!!)	17	60	600	1x
Anti-Salmonella (13)	18	30	600	~50-80 ug/ml
Buffer (TBS!!!)	19	60	600	1x
StrepCy5	20	30	600	5 ug/ml
Buffer (TBS!!!)	21	60	600	1x

Experiment 49a: PDITC slides for iRIf - bBSA/BSA for new device

protocols used :Plasma activation, iRIf slide preparation

2015.08.11

Considerations

prepare 2 PDITC slides for measurement in iRIf

Experiment/Protocol

washed iRIf slides
Plasma activation: 80 L/h, 5 min
APTES incubation: 1 h
PDITC incubation: 5 h
spotted 2 slides (426, 253) with bBSA/BSA for new device

2015.08.12

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA for 5 min
blocked slides with 10 mg/mL BSA for 30 min
washed slides with ddH₂O and dried with wafer gun

Results

Experiment 48: DNA on PDMS

protocols used : PDMS-PDITC, Plasma activation

2015.08.11

Considerations

immobilize Cy5-labeled DNA on PDMS slides

Experiment/Protocol

used 2 flow chamber slides with 3 holes
washed PDMS slides with Ethanol and H₂O
used just 200 µL APTES in Acetone for APTES solution instead of 1 mL APTES in Ethanol
activated slides in Plasma Generator with 40 L/h for 1 min
incubated slides in APTES solution for 2 days+

2015.08.13

each slide was washed with ethanol
a 5 min washing step in Ethanol followed
after drying with wafergun slides were baked in oven for 90 min at 70°C
slides were cooled down with wafergun
incubation in PDITC for 5h40min at RT
filled slideholder with EtOH and discarded it immediately
washed 2x 5 min with EtOH in slideholder
filled slideholder again with EtOH and discarded it immediately
dried with wafergun
1 slide (a) was spotted with 1 µl spots and incubated over night
sample 1 was diluted 1:4,64 (original oncentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 48a:

#	spot	concentration
1	His-tYFP-Spy (lina104) (top 0.5 µl, bottom spot 1 µl)	25 ng/µl
2	His-tYFP-Halo (lina105)	20 ng/µl
3	HA-GFP-His₆-His₆	28.7 ng/µl
4	Control by Tobi: HA-GFP-Halo-His	25 ng/µl

slide (b) was spotted with 3 µl spots and incubated for 2 nights at room temperature
sample 1 was diluted 1:4,64 (original concentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 48b:

#	spot	concentration
1	His-tYFP-Spy (lina104)	25 ng/µl
2	His-tYFP-Halo (lina105)	20 ng/µl
3	HA-GFP-His₆-His₆	28.7 ng/µl
4	Control by Tobi: HA-GFP-Halo-HisHis	25 ng/µl

2015.08.14

Experiment/Protocol

DNA was let dry in
slide 48a was washed with H₂O and dried with wafergun

Results

slide48a was measured in Microarray Scanner for Cy5
no spots could be seen

2015.08.15

Experiment/Protocol

DNA was let dry in
slide 48b was washed with H₂O and dried with wafergun

Results

slide48b was measured in Microarray Scanner for Cy5
no spots could be seen

Experiment 47b: PDITC slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.09

Considerations

prepare 1 PDITC iRIf slides for measurement of antigens, PhyA or bBSA in own device

Experiment/Protocol

prepared 1 PDITC slides
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were stored in slide holder under N₂-atmosphere

2015.08.13

used slides for measurement in own device –> Exp. 52

Experiment 47a: PDITC slides for iRIf - PhyA Testing

protocols used :Plasma activation, iRIf slide preparation

2015.08.09

Considerations

prepare 2 PDITC iRIf slides for measurement of antigens, PhyA or bBSA in own device

Experiment/Protocol

prepared 2 PDITC slides
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were stored in slide holder under N₂-atmosphere

2015.08.10

Experiment/Protocol

spotted 3 µL of different PhyAs and controls on 2 slides

Spotting:

#	spot	Elution no.	Concentration
1-2	PhyA-GFP
3-4	His-PhyA-GST selbst aufgereinigt	2	6.6 mg/ml → 1:10
5	His-PhyA-GST in AntigenSolution (17)	2	1:1 (1:10 His-PhyA-GST in 5mg/ml BSA : AntigenSolution (1:5)
6	GFP (Max)		1 mg/ml
7	GFP-Lysate-NT
8	His-GFP-Halo (Konstrukt von AG-Roth) - selbst aufgereinigt	?
9	bBSA		0.2 mg/ml
10	GFP (Max) in AntigenSolution (17)		1:1 (0.2 mg/ml GFP in 5mg/ml BSA : AntigenSolution (1:5))
11	GFP (Max) in Elutionspuffer		1:1 (0.2 mg/ml GFP in 5mg/ml BSA : Elutionsbuffer)

2015.08.11

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
sildes were blocked with 10 mg/mL BSA for 30 min

Measurement

slide 012 was measured, the other slide is broken and could not be measured.

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	420	1x
anti-phyA (N-term)	4	40	450	5 ug/ml
Buffer	5	60	300	1x
anti-phyA (rabbit, polyclonal)	6	40	450	5 ug/ml
Buffer	7	60	300	1x
anti-GFP (goat, biotinylated)	8	40	450	3 ug/ml
Buffer	9	60	300	1x
anti-GST	10	40	450	5 ug/ml
Buffer	11	60	600	1x
anti-Rabbit (mouse)	12	40	450	5 ug/ml
Buffer	13	60	300	1x
anti-goat	14	40	450	5 ug/ml
Buffer	15	60	300	1x
StrepCy5	16	40	450	5 ug/ml
Buffer	17	60	300	1x

Results

Experiment 46c: Ni-NTA slides for iRIf

protocols used :Plasma activation, iRIf slide preparation

2015.08.09

Considerations

prepare 2 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

Experiment/Protocol

prepared 2 PDITC slides
plasma activation: 80 L/h
APTES incubation: 45 min
PDITC incubation: 2h
slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

2015.08.10

Experiment/Protocol

slides were incubated in blocking solution for 1 h
slides were incubated in Nickel sulfate solution for 1 h
slides were washed 2x in PBS and 1x in diluted PBS
slides were dried and stored under N₂-atmosphere

2015.08.16

Experiment/Protocol

spotted slides 303 and 429 with samples from cell-free
incubated at 4°C o/n

Spotting (Slide 303):

Spot	Protein	Number of spots	Concentration
1-2	BK foil 1	2	unknown
3-4	neg foil 1	2	unknown
5-6	BK 1	2	unknown
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His desalt	1	0.5 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	neg 1	1	unknown

Spotting (Slide 429):

Spot	Protein	Number of spots	Concentration
1-2	BK foil 2	2	unknown
3-4	neg foil	2 2	unknown
5-6	BK 2	2	unknown
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His lysate	1	~0.2 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	neg 2	1	unknown

Results

Slide 429: Selection of ROI

Slide 429: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 429: Binding curves

Slide 303: Selection of ROI

Slide 303: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 303: Binding curves

Experiment 46b: Ni-NTA slides for iRIf - cell free expressed GFP

protocols used :Plasma activation, iRIf slide preparation

2015.08.09

Considerations

prepare 1 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

Experiment/Protocol

prepared 1 PDITC slides
plasma activation: 80 L/h
APTES incubation: 45 min
PDITC incubation: 2h
slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

2015.08.10

Experiment/Protocol

slides were incubated in blocking solution for 1 h
slides were incubated in Nickel sulfate solution for 1 h
slides were washed 2x in PBS and 1x in diluted PBS
slides were dried and stored under N₂-atmosphere

2015.08.13

Experiment/Protocol

spotted cell-free expressed GFP and His-Lysate on slide 208
incubation o/n at 4 °C

Spotting:

#	spot	Concentration
1-3	HA-GFP-2x6His (cellfree expressed))
4-6	neg. control (cellfree Expression without DNA)
7-8	His-GFP Lysate
9	n. T. GFP Lysate)
10	bBSA	0.2 mg/ml
11	Max GFP	0.5 mg/ml

2015.08.14

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
slides were blocked with 10 mg/mL BSA in PBS for 30 min
slides were washed in PBS for 10 min
slides were washed in d1/10 diluted PBS with 20 mM Imidazole
slides were washed in d1/10 diluted PBS

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-GFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
StrepCy5	6	30	600	5 ug/ml
Buffer	7	60	600	1x

Results

Experiment 46a: Ni-NTA slides for iRIf - cell free expressed tYFP

protocols used :Plasma activation, iRIf slide preparation

2015.08.09

Considerations

prepare 2 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

Experiment/Protocol

prepared 2 PDITC slides
plasma activation: 80 L/h
APTES incubation: 45 min
PDITC incubation: 2h
slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

2015.08.10

Experiment/Protocol

slides were incubated in blocking solution for 1 h
slides were incubated in Nickel sulfate solution for 1 h
slides were washed 2x in PBS and 1x in diluted PBS
slides were dried and stored under N₂-atmosphere

spotted slide 254 and 215 with cell free expressed proteins
3 µL of cell free expressed His-tYFP-Halo and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

#	spot	concentration
1	His-tYFP-Halo KK
2	His-tYFP-Halo KK + Mg
3	His-tYFP-Halo BK
4	His-tYFP-Halo BK + Mg
5	His-tYFP-Halo neg.
6	bBSA	200 µg/ml
7	Max His-GFP	0.5 mg/mL
8	His-GFP Lysate	~1 mg/mL
9	His-GFP Lysate	~1 mg/mL
10	Max His-GFP	1 mg/mL
11	nT-GFP Lysate	~1 mg/mL

2015.08.11

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
sildes were blocked with 10 mg/mL BSA for 30 min
slides were washed with PBS for 10 min
slides were washed with 20 mM Imidazole in diluted PBS for 10 min
slides were washed with diluted PBS for 10 min

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-tYFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Results

Experiment 45b: Ni-NTA slides for iRIf - cell free tYFP

protocols used :Plasma activation, iRIf slide preparation

2015.08.07

Considerations

prepare 2 Ni-NTA slides for measurement of cell free expressed proteins and purified antigens in iRIf

Experiment/Protocol

prepared 2 PDITC slides according to protocol
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were incubated with NTA-solution o/n

2015.08.08

Experiment/Protocol

blocking in APTES blocking solution for 1h at RT
NiSO₄ incubation time: 1 h at RT
slides were washed with PBS/diluted PBS

2015.08.09

Experiment/Protocol

used slide 424 and 029
3 µL of cell free expressed His-tYFP-Halo and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

#	spot	concentration
1	His-tYFP-Spy KK
2	His-tYFP-Spy KK + Mg
3	His-tYFP-Spy BK
4	His-tYFP-Spy BK + Mg
5	His-tYFP-Spy neg.
6	bBSA	200 µg/ml
7	Max His-GFP	0.5 mg/mL
8	His-GFP Lysate	~1 mg/mL
9	His-GFP Lysate	~1 mg/mL
10	Max His-GFP	1 mg/mL
11	nT-GFP Lysate	~1 mg/mL

2015.08.10

Experiment/Protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min
slides were washed with PBS, imidazole in diluted PBS and in diluted PBS

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-tYFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Results

Experiment 45a: Ni-NTA slides for iRIf - cell free GFP

protocols used :Plasma activation, iRIf slide preparation

2015.08.07

Considerations

prepare 2 Ni-NTA slides for measurement of cell free expressed proteins
slide 024 and 216

Experiment/Protocol

prepared 2 PDITC slides according to protocol
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were incubated with NTA-solution o/n

2015.08.08

Experiment/Protocol

blocking in APTES blocking solution for 1h at RT
NiSO₄ incubation time: 1 h at RT
slides were washed with PBS/diluted PBS
3 µL of cell free expressed His-GFP and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

#	spot	concentration
1	HA-GFP-His KK
2	HA-GFP-His KK+Mg
3	HA-GFP-His BK
4	HA-GFP-His BK+Mg
5	HA-GFP-His BP
6	HA-GFP-His PP
7	Max His-GFP	0.1 mg/mL
8	nT-GFP Lysate	~1 mg/mL
9	His-GFP Lysate	~1 mg/mL
10	cell free neg.
11	bBSA	100 µg/ml

2015.08.09

Experiment/Protocol

spots were blocked 2x 5 min with 10 mg/mL BSA for 5 min
slides were blocked 10 mg/mL BSA for 30 min

Measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-GFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
StrepCy5	6	30	600	5 ug/ml
Buffer	7	60	600	1x

Results

Experiment 44c: PDITC slides for iRIf - own setup 2

protocols used :Plasma activation, iRIf slide preparation

2015.08.07

Considerations

prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf and bBSA in own setup

Experiment/Protocol

prepared 2 PDITC slides according to protocol
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were stored in desiccator o/n

2015.08.09

Experiment/Protocol

spotted the slides with 200 µg/mL bBSA
9 spots with bBSA, 1 spot with BSA (10 mg/mL) in the middle of the slide
slides were incubated at 4 °C o/n

2015.08.10

Experiment/Protocol

blocked spots 2x with 10 mg/mL BSA for 20 min
blocked slides with 10 mg/mL BSA for 45 min
dried slides with wafergun and stored them under N₂-atmosphere till measurement

Experiment 44b: PDITC slides for iRIf - antigens

protocols used :Plasma activation, iRIf slide preparation

2015.08.07

Considerations

prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf
slide 265 and 426

Experiment/Protocol

prepared 2 PDITC slides according to protocol
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were stored in desiccator o/n

2015.08.08

Experiment/Protocol

antigens were diluted to a concentration of ~0.4-0.5 mg/mL
3µL of antigens and controls (GFP, bBSA) were spotted and icubated o/n
spinned antigens before spotting

Spotting pattern:

#	spot	Concentration
1-2	HIV(17)	0.4-0.5 mg/ml
3-4	HCV(10)	0.4-0.5 mg/ml
5-6	Tetanus(11)	0.4-0.5 mg/ml
7	GFP	0.1 mg/ml
8	PhyA	0.4-0.5 mg/ml
9	bBSA	0.1 mg/ml
10-11	Salmonella(15)	0.4-0.5 mg/ml

2015.08.09

Experiment/Protocol

Slide 265 - measured in new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
Anti-HIV (rabbit,pk))	4	45	600	10 ug/ml
Buffer	5	45	600	1x
Anti-HCV (rabbit,pk)	6	45	600	10 ug/ml
Buffer	7	45	600	1x
Anti-phyA (rabbit,N-term.)	8	45	600	10 ug/ml
Buffer	9	45	600	1x
Anti-Rabbit	10	30	600	5 ug/ml
Buffer	11	45	600	1x
Anti-HIV (mouse, monoklonal)	12	45	600	10 ug/ml
Buffer	13	45	600	1x
Anti-HCV (mouse, monoklonal)	14	45	600	10 ug/ml
Buffer	15	45	600	1x
Anti-Tetanus (mouse, monoklonal)	16	45	600	10 mg/ml
Buffer	17	45	600	1x
Anti-GFP (goat)	18	30	600	5 ug/ml
Buffer	19	45	600	1x
StrepCy	20	45	600	5 ug/ml
Buffer	21	45	600	1x
Anti-Mouse	22	30	600	5 ug/ml
Buffer	23	45	600	1x

Experiment 44a: PDITC slides for iRIf - own setup 1

protocols used :Plasma activation, iRIf slide preparation

2015.08.07

Considerations

prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf and bBSA in own setup

Experiment/Protocol

prepared 2 PDITC slides according to protocol
plasma activation: 80 L/h
APTES incubation: 30 min
PDITC incubation: 2 h
slides were stored in desiccator o/n

2015.08.08

Experiment/Protocol

spotted the slides with 200 µg/mL bBSA
9 spots with bBSA, 1 spot with BSA (10 mg/mL) in the middle of the slide
slides were incubated at 4 °C for 11 h
slides were blocked and afterwards measured in own setup

Results

Experiment 43: Ni/NTA slides for Normann

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 2 iRIf slides from Normann (309, 310)

2015.08.04

Experiment/Protocol

6 iRIf slides were cleaned according to iRIf slide wash steps
plasma activation for 5 min at 80 L/h gasflow
incubated in APTES solution for 30 min
incubation in PDITC solution for 2.5 h
sandwiched slides with 80 µL NTA and incubated them at 4°C o/n

2015.08.05

Experiment/Protocol

blocked slides for 1 h in APTES blocking solution
washed 2x in PBS for 10 min
incubated in NiSO₄-solution for 1 h
washed 1x in PBS for 10 min
washed 2x in diluted PBS for 10 min then dried with wafer gun
stored slides in slideholder under N₂-atmosphere for Norman (uses them the next day)

Results

Experiment 42c: washed iRIf slides for Halo-surface (Piehler) with Günter

protocols used :iRIf slide wash steps

Considerations

wash six of Jürgens iRIf slides (254, 466, 423, 426, 253 ,424) for first test with Halo

2015.08.05

Experiment/Protocol

2 slides: 423, 466
slides were cleaned with acetone, isopropanol and aqua dest
plasma activation for 10 min at gas flow 60 L/h with valve 2 (air)
slides were cross-sandwiched (Piehler style) with 15 µL of PLL-PEG-HTL for 15 min
3 µL of Halo-GFP-His, Halo-mCherry-His, bBSA, and 2 µL of pos./neg. controls (from Piehler: His-Halo-GFP/His-GFP) were spotted

spotting pattern:

#	spot	concentration
1-2	Halo-GFP 1:20
3-4	Halo-GFP 1:10
5-6	Halo-mCHERRY 1:20
7	His-Halo-GFP Piehler
8	His-GFP Piehler
9	bBSA	100 µg/ml
10-11	Halo-mCHERRY 1:10

spots were blocked 2x with 10 mg/mL for 5 min
slides were blocked in 10 mg/ml BSA for 30 min
slide were washed with ddH₂O

Measurement

Slide 423 - measured in new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-His (from AG Roth)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Slide 466 - measured in old setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-His (from iGEM Lab)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Cy5 Fluorescence was measured afterwards

Results

Slide 423: Selection of ROI

Slide 423: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 423: Binding curves

Slide 466: Selection of ROI

Slide 466: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 466: Binding curves

→ plasmaactivation with pure air does not lead to better binding of PLL-PEG-HTL to the surface

Experiment 42b: washed iRIf slides for Halo-surface (Piehler) with Günter

protocols used :iRIf slide wash steps

Considerations

wash six of Jürgens iRIf slides (254, 466, 423, 426, 253 ,424) for first test with Halo

2015.08.05

Experiment/Protocol

2 slides: 426, 424
slides were cleaned with acetone, isopropanol and aqua dest
plasma activation for 10 min at gas flow 60 L/h with valve 1 (steam)
slides were cross-sandwiched (Piehler style) with 15 µL of PLL-PEG-HTL for 15 min
3 µL of Halo-GFP-His, Halo-mCherry-His, bBSA, and 2 µL of pos./neg. controls (from Piehler: His-Halo-GFP/His-GFP) were spotted

spotting pattern:

#	spot	concentration
1-2	Halo-GFP 1:20
3-4	Halo-GFP 1:10
5-6	Halo-mCHERRY 1:20
7	His-Halo-GFP Piehler
8	His-GFP Piehler
9	bBSA	100 µg/ml
10-11	Halo-mCHERRY 1:10

slide 426

slide wasn't blocked at all → measured directly

slide 424

spots were blocked 2x with 10 mg/mL for 5 min
slide was blocked in 10 mg/ml BSA for 30 min
slide was washed with ddH₂O

Measurement

Slide 426 - measured in old setup

slide wasn't blocked before measurement

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-GFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
StrepCy5	6	30	600	5 ug/ml
Buffer	7	60	600	1x

Slide 424 - measured in old setup (Strep-Cy5 and a-GFP step were accidently switched)

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-His	4	30	600	5 ug/ml
Buffer	5	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x

Cy5 Fluorescence was measured afterwards

Results

Slide 424: Selection of ROI

Slide 424: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 424: Binding curves

Slide 426: Selection of ROI

Slide 426: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 426: Binding curves

for slide 426: during the BSA step lots of BSA bound to the surface –> should block before

Experiment 42a: washed iRIf slides for Halo-surface (Silan by abcr) with Günter

protocols used :iRIf slide wash steps

Considerations

wash six of Jürgens iRIf slides (254, 466, 423, 426, 253 ,424) for first test with Halo

2015.08.05

Experiment/Protocol

2 slides: 254, 253
slides were cleaned with acetone, isopropanol and aqua dest
plasma activation for 10 min at gas flow 60 L/h with valve 1 (steam)
slides were sandwiched with 80 µL of abcr-silane and incubated for 1 h
washed 30s in acetone
dried with N₂
3 µl of Halo-GFP, Halo-mCHERRY and bBSA as well as 2 µl of His-Halo-GFP (Piehler positive control) and His-GFP (Piehler negative control) were spotted

spotting pattern:

#	spot	concentration
1-2	Halo-GFP 1:20
3-4	Halo-GFP 1:10
5-6	Halo-mCHERRY 1:20
7	His-Halo-GFP Piehler
8	His-GFP Piehler
9	bBSA	100 µg/ml
10-11	Halo-mCHERRY 1:10

spots were blocked 2x with 10 mg/mL for 5 min
slides were blocked in 10 mg/ml BSA for 30 min
slide were washed with ddH₂O

Measurement

slide 254 - measured with new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-His (from AG Roth)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

slide 253 - measured with new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-His (from AG Roth)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Cy5 Fluorescence was measured afterwards

Results

Slide 253: Selection of ROI

Slide 253: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 253: Binding curves

Slide 253: Selection of ROI

Slide 253: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 253: Binding curves

Experiment 41b: PDITC iRIf slides for Halo-surface (Wiesmüller Halo 2) with Günter

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 6 of our iRIf slides with APTES/PDITC surface for first test with Halo

2015.08.04

Experiment/Protocol

6 iRIf slides were cleaned according to iRIf slide wash steps
plasma activation for 5 min at 80 L/h gasflow
incubated in APTES solution for 40 min
incubation in PDITC solution for 2.5 h
slides were stored in desiccator o/n

2015.08.05

Experiment/Protocol

3 slides: 287, 215, 315
slides were incubated in slideholder with Halo 2-solution for 2 days

2015.08.07

Experiment/Protocol

3 µL of cell free expressed proteins, Halo-GFP, Halo-mCherry and pos/neg control (from Piehler) were spotted on slides 215 and 315
slides were incubated for 1 h at RT

spotting pattern:

#	spot	concentration
1-2	Koko pos.
3-4	Koko neg.
5-6	Promega pos.
7	Halo-GFP (pos, Piehler)
8	His-GFP (neg, Piehler)
9	bBSA	100 µg/ml
10	Halo-GFP 1:10
11	Halo-mCHERRY 1:10

slide 287 was first blocked with 10 mg/mL BSA for 30 min
then 3 µL of Halo-GFP, His-GFP, pos/neg control (from Piehler), His-GFP and mCherry were spotted on slide 287
slide was incubated for 1 h at RT

spotting pattern:

#	spot	concentration
1-3	Halo-GFP
4-6	Halo-mCherry
7	Halo-GFP (pos, Piehler)
8	His-GFP (neg, Piehler)
9	bBSA	100 µg/ml
10	His-GFP
11	His-mCHERRY

for all 3 slides:

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min

Measurement

slide 215 and 315

slide 215 was measured in old setup, slide 315 was measured in new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-tYFP	4	30	600	5 µg/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 µg/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 µg/ml
Buffer	9	60	600	1x

slide 287 was measured with the following flush protocol in the new setup

Reagent	Step	Flow rate (µl/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 µg/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 µg/ml
Buffer	9	60	600	1x

Cy5 Fluorescence was measured afterwards

Results

Slide 215: Selection of ROI

Slide 215: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 287: Selection of ROI

Slide 287: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 315: Selection of ROI

Experiment 41a: PDITC iRIf slides for Halo-surface (Wiesmüller Halo 1) with Günter

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 6 of our iRIf slides with APTES/PDITC surface for first test with Halo

2015.08.04

Experiment/Protocol

6 iRIf slides were cleaned according to iRIf slide wash steps
plasma activation for 5 min at 80 L/h gasflow
incubated in APTES solution for 40 min
incubation in PDITC solution for 2.5 h
slides were stored in desiccator o/n

2015.08.05

Experiment/Protocol

3 slides: 012, 208, 121
slides were incubated in slideholder with Halo 1-solution for 2 days

2015.08.07

Experiment/Protocol

3 µL of cell free expressed proteins, Halo-GFP, Halo-mCherry and pos/neg control (from Piehler) were spotted on slides 208 and 121
slides were incubated for 1 h at RT

spotting pattern:

#	spot	concentration
1-2	Koko pos.
3-4	Koko neg.
5-6	Promega pos.
7	Halo-GFP (pos, Piehler)
8	His-GFP (neg, Piehler)
9	bBSA	100 µg/ml
10	Halo-GFP 1:10
11	Halo-mCHERRY 1:10

slide 012 was first blocked with 10 mg/mL BSA for 30 min
then 3 µL of Halo-GFP, His-GFP, pos/neg control (from Piehler), His-GFP and mCherry were spotted on slide 012
slide was incubated for 1 h at RT

spotting pattern:

#	spot	concentration
1-3	Halo-GFP
4-6	Halo-mCherry
7	Halo-GFP (pos, Piehler)
8	His-GFP (neg, Piehler)
9	bBSA	100 µg/ml
10	His-GFP
11	His-mCHERRY

for all 3 slides:

spots were blocked 2x with 10 mg/mL BSA for 5 min
slides were blocked with 10 mg/mL BSA for 30 min

Measurement

slide 208 and 121 slide 208 was measured in old setup, slide 121 was measured in new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
a-tYFP	4	30	600	5 µg/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 µg/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 µg/ml
Buffer	9	60	600	1x

slide 012 was measured with the following flush protocol in the new setup

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 µg/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 µg/ml
Buffer	9	60	600	1x

Cy5 Fluorescence was measured afterwards

Results

Slide 121: Selection of ROI

Slide 121: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 208: Selection of ROI

Slide 208: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

Slide 012: Selection of ROI

Slide 012: Picture obtained from fluorescence scanner. Top: Emission with 635 nm; Bottom Emission with 523 nm

→ Halo surface is not homogenous enough

→ strong unspecific binding occuring

Experiment 40b: PDITC iRIf slides for measurement in own setup

protocols used :Plasma activation, iRIf slide preparation

Considerations

prepare 4 iRIf PDTIC slides for measurement
store slides for future measurements
slides were used for measurement in own setup

2015.08.03

Experiment/Protocol

4 PDITC iRIf slides were prepared according to protocol
plasma activation for 5 min at 80 L/h
slides were incubated in APTES for 45 min
slides were incubated in PDITC for 2 h, washed with Ethanol and Acetone and stored in the desiccator for 40 min
slides were stored at 4 °C under N₂-atmosphere

2015.08.04

Experiment/Protocol

3 µL of bBSA and BSA were spotted on the slides for measurement in own setup

spotting pattern:

#	spot
4+5	bBSA 500 µg/mL
6+10	bBSA 200 µg/mL
11	BSA 10 mg/mL

slides were incubated at 4 °C o/n

Experiment 40a: PDITC iRIf slides

protocols used :Plasma activation, [[protocols:irif_slide_preparation|iRIf slide preparation]]

Considerations

prepare 4 iRIf PDTIC slides for measurement
spot antibodies that shall be detected with antigens (opposite to Exp 37a) on 2 slides

2015.08.03

Experiment/Protocol

4 PDITC iRIf slides were prepared according to protocol
plasma activation for 5 min at 80 L/h
slides were incubated in APTES for 45 min
slides were incubated in PDITC for 2 h, washed with Ethanol and Acetone and stored in the desiccator for 40 min
3µL of the Antibodies for HIV, HCV, Tetanus and Salmonella + controls (GFP, bBSA) were spotted

spotting pattern:

#	spot (AB)	AB-Concentration [µg/mL]	Antigen	Elution no.	Antigenconc.
1-2	gp41 DDX1306	100	HIV(17)	1	1.0 mg/mL??
3-4	HCV-AB	100	HCV(10)	1	0.74 mg/mL??
5-6	HYB 278-01	100	Tetanus(11)	1	3.75 mg/mL??
7-8	GFP	1.0 mg/mL	a-GFP (goat;biotinylated)		5
9	bBSA	0.1 mg/mL	Strep-Cy5		(strep-Cy5) 10
10-11	a-Salmonella-(pIG15_1301)	100	Salmonella(15)	2	6.7 mg/mL

slides were incubated at 4°C o/n

2015.08.04

Experiment/protocol

spots were blocked 2x with 10 mg/mL BSA for 5 min
slide was blocked in 10 mg/mL in BSA for 30 min
slides were washed 1x with ddH₂O
slides were measured in iRIf

Measurement

Only 1 slide was measured. Second slide syringe didnt suck solutions.

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	940	1x
BSA	2	30	900	10 mg/ml
Buffer	3	30	600	1x
HIV (17)	4	20	900	0.25 mg/ml
Buffer	5	30	600	1x
HCV (10)	6	20	900	0.25 mg/ml
Buffer	7	30	600	1x
Tetanus (11)	8	20	900	0.25 mg/ml
Buffer	9	30	600	1x
Anti-Mouse	10	20	900	5 ug/ml
Buffer	11	30	600	1x
Anti-GFP	12	20	900	3 ug/ml
Buffer	13	30	600	1x

Results

Antigens didn't bind to the immobilized antibodies, anti-mouse only bound to a-Tetanus spot, and not to a-HIV and a-HCV spots, even though they are from mice, too.

Slide 29: Selection of ROIs

Slide 29: Binding curve

Team:Freiburg/Labjournals/SurChem/August

Labjournal Surchem August

Experiment 63e: Anti Mouse/Rabbit on PDITC

2015.08.29

Experiment/Protocol

2015.08.31

Experiment/Protocol

Results

Experiment 63d: Stockholm II

2015.08.29

Experiment/Protocol

2015.08.30

Experiment/Protocol

2015.08.31

Experiment/Protocol

Results

Experiment 63c: Tetanus on PDITC

2015.08.29

Experiment/Protocol

2015.08.30

Experiment/Protocol

2015.08.31

Experiment/Protocol

Results

Experiment 63b: Ni-NTA iRIf slides

2015.08.29

Experiment/Protocol

2015.08.30

Experiment/Protocol

Experiment 63a: Salmonella antigene on PDITC in iRIf

2015.08.29

Experiment/Protocol

2015.08.30

Experiment/Protocol

Results

Experiment 62c: PDITC/Ni-NTA slides for iRIf

2015.08.26

Experiment/Protocol

Experiment 62b:Tetanus antigen on PDITC slides for iRIf

2015.08.26

Experiment/Protocol

2015.08.29

Experiment/Protocol

2015.08.30

Experiment/Protocol

Results

Experiment 62a: PDITC/Ni-NTA slides for iRIf - Stockholm collaboration

2015.08.26

Experiment/Protocol

2015.08.27

Experiment/Protocol

2015.08.29

Experiment/Protocol

Results

Experiment 61: DNA on PDMS 6.0

Considerations

2015.08.25

Experiment/Protocol

2015.08.26

Experiment/Protocol

2015.08.27

Experiment/Protocol

Results

2015.08.28

Measurement

Results

Experiment 60: DNA on PDMS 5.0

Considerations

2015.08.23

Experiment/Protocol

2015.08.24

2015.08.25

Experiment/Protocol

Results

2015.08.26

Results

Experiment 59d on slide cellfree expressed GFP on Ni-NTA slides for iRIf

2015.08.21

Experiment/Protocol

2015.08.25