Team:Freiburg/Labjournals/SurChem/August

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Labjournal Surchem August

Experiment 63e: Anti Mouse/Rabbit on PDITC

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306003 ug/ml
Buffer5603001x
anti- rabbit 630600
Buffer7603001x
anti- mouse 830600
Buffer9603001x

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

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/Fc100 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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306003 ug/ml
Buffer5603001x
affibody 620900diluted 1:3
Buffer7603001x
affibody820900undiluted
Buffer9603001x
Anti His1030600undiluted
Buffer11603001x

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306003 ug/ml
Buffer5603001x
Serum (-/+) Sig002630900diluted in 5 mg/ml BSA in PBS
Buffer7603001x

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

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306003 ug/ml
Buffer5603001x
anti-Salmonella (1301, desalt)6309001:2 Dilution in 5 mg/ml BSA in PBS
Buffer7603001x

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

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306003 ug/ml
Buffer5603001x
Serum (-/+) Sig0026309001:10 diluted in 5 mg/ml BSA in PBS
Buffer7603001x

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

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

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
1HA-GFP-Halo-His-His (Tobi)25 ng/µl3 µl
2HA-GFP-Halo-His-His (Tobi)25 ng/µl5 µl
3Cy3-HA-GFP-His-His33 ng/µl4 µl
4Cy3-HA-GFP-His-His66.1 ng/µl4 µl

spotting pattern (slide 61b):

# sample concentration spot size
1Cy3-HA-GFP-His-His66.1 ng/µl5 µl
2HA-GFP-Halo-His-His (Tobi)99 ng/µl3 µl
3Cy3-HA-GFP-His-His66.1 ng/µl10 µl

spotting pattern (slide 61c):

# sample concentration spot size
1Cy3-HA-GFP-His-His66.1 ng/µl3 µl
2Cy3-HA-GFP-His-His33 ng/µl4 µl
3HA-GFP-Halo-His-His (Tobi)25 ng/µl3 µl
4HA-GFP-Halo-His-His (Tobi)25 ng/µl5 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
Buffer1604501x
BSA26045010 mg/ml
Buffer3604501x
anti-GFP4306003 ug/ml
Buffer5603001x
strep-cy56603005 ug/ml
Buffer7603001x

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

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
1HA-GFP-Halo-His-His (Tobi)25 ng/µl1 µl
2Cy5-HA-GFP-His-His12.5 ng/µl3 µl
3Cy5-HA-GFP-His-His44.8 ng/µl1 µl
4Cy5-HA-GFP-His-His28.7 ng/µl0.5 µl
5HA-GFP-Halo-His-His (Tobi)50 ng/µl1 µl
6Cy5-HA-GFP-His-His17.9 ng/µl2 µl
7Cy5-HA-GFP-His-His22.4 ng/µl3 µl
8Cy5-HA-GFP-His-His44.8 ng/µl0.2 µl
9HA-GFP-Halo-His-His (Tobi)99 ng/µl1 µl

spotting pattern (slide 60b):

# sample concentration spot size
1HA-GFP-Halo-His-His (Tobi)99 ng/µl3 µl
2HA-GFP-Halo-His-His (Tobi)50 ng/µl3 µl
3PCR-Mix von Cy5-HA-GFP-His-His (44.8 ng/µl)-14 µl
4HA-GFP-Halo-His-His (Tobi)25 ng/µl3 µl
5Cy5-HA-GFP-His-His22.4 ng/µl6 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

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
Buffer1604501x
BSA26045010 mg/ml
Buffer3604501x
Anti-GFP (goat, biotinylated)4306003 ug/ml
Buffer5603001x
StrepCy56303005 ug/ml
Buffer7603001x

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

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
Buffer1604501x
BSA26045010 mg/ml
Buffer3604501x
Anti-GFP (goat, biotinylated)4306003 ug/ml
Buffer5603001x
StrepCy56303005 ug/ml
Buffer7603001x

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

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)1604501x
BSA 26045010 mg/ml
Buffer (PBS)3604501x
Anti-Tetanus (goat, pk)43060025 ug/ml
Buffer (PBS)5603001x
Anti-GFP (biotinylated, Goat)6306003 ug/ml
Buffer (PBS)7603001x
Anti-Goat8306005 ug/ml
Buffer (PBS)9603001x
StrepCy510303005 ug/ml
Buffer (PBS)11603001x

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

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)1607201x
BSA 26090010 mg/ml
Buffer (PBS)3606001x
Serum rabbit Anti-GFP4306001:330
Buffer (PBS)5603001x
Serum (+) SIG0026306001:10
Buffer (PBS)7603001x
Anti-Human8603001 ug/ml
Buffer (PBS)9603001x

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)1607201x
BSA 26090010 mg/ml
Buffer (PBS)3606001x
Serum (-) SIG0024306001:10
Buffer (PBS)5603001x
Serum (+) SIG0026306001:10
Buffer (PBS)7603001x
Anti-Human8603001 ug/ml
Buffer (PBS)9603001x
Anti-GFP (biotinylated, Goat)10603001 ug/ml
Buffer (PBS)11603001x
Strep-Cy512603005 ug/ml
Buffer (PBS)13603001x

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

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)1607201x
His-HIV-Lysate (17)/BSA 2609001:1
Buffer (TBS)3606001x
Anti-GFP (biotinylated, goat)4306005 ug/ml
Buffer (TBS)5603001x
StrepCy56603005 ug/ml
Buffer (TBS)7603001x
Anti-Salmonella desalt (13)830600~60 µg/mL
Buffer (TBS)9603001x
Anti-Salmonella Lysate (13)1030600~60 µg/mL
Buffer (TBS)11603001x

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

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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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

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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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
Buffer1607801x
BSA26078010 mg/ml
Buffer3606001x
Serum (-)430600 *
Buffer5603001x
Serum (+)/a-GFP630600 * / 3ug/ml
Buffer7606001x
anti-Rabbit8306005 ug/ml
Buffer9606001x
Strep Cy5103060005 ug/ml
Buffer11606001x

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

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
1Cy5-HA-GFP-His-His12.5 ng/µl
2Cy5-HA-GFP-His-His17.9 ng/µl
3His-tYFP-Spy (lina104)25 ng/µl
4HA-GFP-Halo-His-His (Tobi)25 ng/µl
5Cy5-HA-GFP-His-His22.4 ng/µl
6PCR-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

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
1Cy5-HA-GFP-His-His22,4 ng/µl
2Control:HA-GFP-Halo-His-His25 ng/µl
3PCR-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

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
Buffer1607801x
BSA26078010 mg/ml
Buffer3606001x
Serum (-)430600 *
Buffer5603001x
Serum (+)/a-GFP630600 * / 3ug/ml
Buffer7606001x
anti-Rabbit8306005 ug/ml
Buffer9606001x
Strep Cy5103060005 ug/ml
Buffer11606001x

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

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

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 GFP0.02 mg/ml
7 bBSA0.2 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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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

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 GFP0.02 mg/ml
7 bBSA0.2 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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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

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

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 GFP0.02 mg/ml
7 bBSA0.2 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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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

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 GFP0.02 mg/ml
7 bBSA0.2 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)1607201x
BSA 26072010 mg/ml
Buffer (PBS)3606001x
Anti GFP (biotinylated, goat)4306005 ug/ml
Buffer (PBS)5603001x
StrepCy56603005 ug/ml
Buffer (PBS)7603001x

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

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

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
1His-tYFP-Spy (lina104) (top 0.5 µl, bottom spot 1 µl)25 ng/µl
2His-tYFP-Halo (lina105)20 ng/µl
3HA-GFP-His6-His628.7 ng/µl
4Control by Tobi: HA-GFP-Halo-His25 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
1His-tYFP-Spy (lina104)25 ng/µl
2His-tYFP-Halo (lina105)20 ng/µl
3HA-GFP-His6-His628.7 ng/µl
4Control by Tobi: HA-GFP-Halo-HisHis25 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

532 nm 635 nm

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.

532 nm 635 nm

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

51a 51b

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

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
Buffer1607801x
BSA26078010 mg/ml
Buffer3606001x
Serum (-)430600 *
Buffer5603001x
Serum (+)/a-GFP630600 * / 3ug/ml
Buffer7606001x
anti-Rabbit8306005 ug/ml
Buffer9606001x
Strep Cy5103060005 ug/ml
Buffer11606001x

Results

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

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!!!)1607201x
BSA 26060010 mg/ml
Buffer (TBS!!!)3606001x
Anti-HIV (polyclonal,rabbit)43060010 ug/ml
Buffer (TBS!!!)5606001x
Anti-HCV (polyclonal,rabbit)63060010 ug/ml
Buffer (TBS!!!)7606001x
Anti-Rabbit (from mouse)8306005 ug/ml
Buffer (TBS!!!)9606001x
Anti-HIV (monoclonal,mouse)103060010 ug/ml
Buffer (TBS!!!)11606001x
Anti-HCV (monoclonal,mouse)123060010 ug/ml
Buffer (TBS!!!)13606001x
Anti-His14306005 ug/ml
Buffer (TBS!!!)15606001x
Anti-GFP (biotinylated, goat)16306003 ug/ml
Buffer (TBS!!!)17606001x
StrepCy518306005 ug/ml
Buffer (TBS!!!)19606001x

Results

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

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!!!)1607201x
BSA 26060010 mg/ml
Buffer (TBS!!!)3606001x
Anti-HIV (polyclonal,rabbit)43060010 ug/ml
Buffer (TBS!!!)5606001x
Anti-HCV (polyclonal,rabbit)63060010 ug/ml
Buffer (TBS!!!)7606001x
Anti-Rabbit (from mouse)8306005 ug/ml
Buffer (TBS!!!)9606001x
Anti-HIV (monoclonal,mouse)103060010 ug/ml
Buffer (TBS!!!)11606001x
Anti-HCV (monoclonal,mouse)123060010 ug/ml
Buffer (TBS!!!)13606001x
Anti-Mouse14306005 ug/ml
Buffer (TBS!!!)15606001x
Anti-GFP (biotinylated, goat)16306003 ug/ml
Buffer (TBS!!!)17606001x
StrepCy518306005 ug/ml
Buffer (TBS!!!)19606001x

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

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

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!!!)1607201x
BSA 26060010 mg/ml
Buffer (TBS!!!)3606001x
Anti-HIV (polyclonal,rabbit)43060010 ug/ml
Buffer (TBS!!!)5606001x
Anti-HCV (polyclonal,rabbit)63060010 ug/ml
Buffer (TBS!!!)7603001x
Anti-Rabbit (from mouse)8306005 ug/ml
Buffer (TBS!!!)9606001x
Anti-HIV (monoclonal,mouse)103060010 ug/ml
Buffer (TBS!!!)11606001x
Anti-HCV (monoclonal,mouse)123060010 ug/ml
Buffer (TBS!!!)13606001x
Anti-Mouse14306005 ug/ml
Buffer (TBS!!!)15606001x
Anti-GFP (biotinylated, goat)16306003 ug/ml
Buffer (TBS!!!)17606001x
Anti-Salmonella (13)1830600~50-80 ug/ml
Buffer (TBS!!!)19606001x
StrepCy520306005 ug/ml
Buffer (TBS!!!)21606001x

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

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

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
1His-tYFP-Spy (lina104) (top 0.5 µl, bottom spot 1 µl)25 ng/µl
2His-tYFP-Halo (lina105)20 ng/µl
3HA-GFP-His6-His628.7 ng/µl
4Control by Tobi: HA-GFP-Halo-His25 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
1His-tYFP-Spy (lina104)25 ng/µl
2His-tYFP-Halo (lina105)20 ng/µl
3HA-GFP-His6-His628.7 ng/µl
4Control by Tobi: HA-GFP-Halo-HisHis25 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

532 nm 635 nm

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

532 nm 635 nm

Experiment 47b: PDITC slides for iRIf

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

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
Buffer1607801x
BSA26060010 mg/ml
Buffer3604201x
anti-phyA (N-term)4404505 ug/ml
Buffer5603001x
anti-phyA (rabbit, polyclonal)6404505 ug/ml
Buffer7603001x
anti-GFP (goat, biotinylated)8404503 ug/ml
Buffer9603001x
anti-GST10404505 ug/ml
Buffer11606001x
anti-Rabbit (mouse)12404505 ug/ml
Buffer13603001x
anti-goat14404505 ug/ml
Buffer15603001x
StrepCy516404505 ug/ml
Buffer17603001x

Results

Experiment 46c: Ni-NTA slides for iRIf

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-GFP4306003 ug/ml
Buffer5306001x
StrepCy56306005 ug/ml
Buffer7606001x

Results

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-tYFP4306003 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy58306005 ug/ml
Buffer9606001x

Results

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-tYFP4306003 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy58306005 ug/ml
Buffer9606001x

Results

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-GFP4306003 ug/ml
Buffer5306001x
StrepCy56306005 ug/ml
Buffer7606001x

Results

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

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

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 Elution no. 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
Buffer1607201x
BSA26060010 mg/ml
Buffer3606001x
Anti-HIV (rabbit,pk))44560010 ug/ml
Buffer5456001x
Anti-HCV (rabbit,pk)64560010 ug/ml
Buffer7456001x
Anti-phyA (rabbit,N-term.)84560010 ug/ml
Buffer9456001x
Anti-Rabbit10306005 ug/ml
Buffer11456001x
Anti-HIV (mouse, monoklonal)124560010 ug/ml
Buffer13456001x
Anti-HCV (mouse, monoklonal)144560010 ug/ml
Buffer15456001x
Anti-Tetanus (mouse, monoklonal)164560010 mg/ml
Buffer17456001x
Anti-GFP (goat)18306005 ug/ml
Buffer19456001x
StrepCy20456005 ug/ml
Buffer21456001x
Anti-Mouse22306005 ug/ml
Buffer23456001x

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

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

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-His (from AG Roth)4306005 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy8306005 ug/ml
Buffer9606001x

Slide 466 - measured in old setup

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-His (from iGEM Lab)4306005 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy8306005 ug/ml
Buffer9606001x
  • 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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-GFP4306003 ug/ml
Buffer5306001x
StrepCy56306005 ug/ml
Buffer7606001x

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

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-His4306005 ug/ml
Buffer5306001x
StrepCy8306005 ug/ml
Buffer9606001x
a-GFP6306003 ug/ml
Buffer7306001x
  • 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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-His (from AG Roth)4306005 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy8306005 ug/ml
Buffer9606001x

slide 253 - measured with new setup

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-His (from AG Roth)4306005 ug/ml
Buffer5306001x
a-GFP6306003 ug/ml
Buffer7306001x
StrepCy8306005 ug/ml
Buffer9606001x
  • 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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-tYFP4306005 µg/ml
Buffer5306001x
a-GFP6306003 µg/ml
Buffer7306001x
StrepCy8306005 µg/ml
Buffer9606001x

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

Reagent Step Flow rate (µl/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer5306001x
a-GFP6306003 µg/ml
Buffer7306001x
StrepCy8306005 µg/ml
Buffer9606001x
  • 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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
a-tYFP4306005 µg/ml
Buffer5306001x
a-GFP6306003 µg/ml
Buffer7306001x
StrepCy8306005 µg/ml
Buffer9606001x

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

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer5306001x
a-GFP6306003 µg/ml
Buffer7306001x
StrepCy8306005 µg/ml
Buffer9606001x
  • 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

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

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
Buffer1609401x
BSA23090010 mg/ml
Buffer3306001x
HIV (17)4209000.25 mg/ml
Buffer5306001x
HCV (10)6209000.25 mg/ml
Buffer7306001x
Tetanus (11)8209000.25 mg/ml
Buffer9306001x
Anti-Mouse10209005 ug/ml
Buffer11306001x
Anti-GFP12209003 ug/ml
Buffer13306001x

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