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Labjournal Surchem July

Experiment 39b: PDITC iRIf slides for measurement with PhyA

protocols used :iRIf slide preparation

Considerations

preparing 4 iRIf PDITC slides for measurement
spot PhyA on 2 slides

2015.07.30

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 30 min
slides were incubated in PDITC for 2 h
slides were stored o/n in desiccator

2015.07.31

Experiment/protocol

for storage until Sunday 02.08.2015 the slides were taken out of the desiccator and put in a slideholder under N₂-atmosphere at 4 °C

2015.08.02

3 µL of samples (1-3,6) and controls (GFP,bBSA) were spotted and incubated o/n at 4 °C. For samples 4-5 and 10-11 4 µl were spotted.

spotting pattern:

#	spot	Elution no.	Concentration	Antibody	AB-conc.[µg/ml]
1-2	Phy A	2	6.6 mg/ml		5 µg/ml
3	Phy A	3	2.3. mg/ml		5 µg/ml
4-5	Promega 104			anti-tYFP	20
6	rabbit AB (anti-HCV)		1 mg/mL	anti-rabbit	20
7	Max GFP		1.0 mg/mL	a-GFP (goat;biotinylated)	5
8	His-GFP Lysate			a-GFP (goat;biotinylated)	5
9	bBSA		0.1 mg/mL	Strep-Cy5	10
10-11	Promega 104 + feed			anti-tYFP	20
				anti-His (mouse)	20

2015.08.03

Experiment/Protocol

protein solution was removed with pipette and spots were blocked 2x 5 min with BSA 10 mg/ml in PBS
slides were blocked in 10 mg/mL BSA in PBS for 30 min
slides were washed in PBS for 10 min
slides were washed 2x in diluted PBS 1/10 for 10 min
slides were dried with wafer gun and measured in iRIf

Measurement

Slide 1

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	45	600	10 mg/ml
Buffer	3	60	600	1x
Anti-GFP(goat;biotinylated)	4	25	720	5 ug/ml
Buffer	5	60	300	1x
Anti-tYFP (rabbit)	6	15	1200	20 ug/ml
Buffer	7	60	300	1x
Anti-PhyA (rabbit)	8	20	900	20 ug/ml
Buffer	9	60	300	1x
Anti-Rabbit	10	30	600	5 ug/ml
Buffer	11	60	300	1x
StrepCy5	12	30	600	5 ug/ml
Buffer	13	60	300	1x

slide 2

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	45	500	10 mg/ml
Buffer	3	60	600	1x
Anti-GFP(goat;biotinylated)	4	25	720	5 ug/ml
Buffer	5	60	300	1x
Anti-tYFP (rabbit)	6	15	1200	20 ug/ml
Buffer	7	60	300	1x
Anti-PhyA (rabbit, N-terminal)	8	20	900	5 ug/ml
Buffer	9	60	300	1x
Anti-Rabbit	10	30	600	15 ug/ml
Buffer	11	60	300	1x
StrepCy5	12	30	600	5 ug/ml
Buffer	13	60	300	1x

another anti-Phy was used, because the one which was used in the first measurement was empty and also Phillip told us, that this one should bind better

Results

Binding of anti-phyA to phyA was not observed in RIfs
much of the protein was washed away from the spotted spot.
- We have to take lower concentrations for spotting (100-400 ug/ml)
no Anti-tYFP to tYFP binding could be detected
- to few tYFP on the slides because all proteins in Lysate bind to PDITC
- should use specific surface (e.g. Ni-NTA, Halo) next time, where only tYFP binds

Slide 303: ROI selection

Slide 303: binding curves

Slide 303: Inverted QP during initial buffer step: Shows that already much of the PhyA is flushed away from the spot

Experiment 39a: PDITC iRIf slides for measurement with purified antigens

protocols used :iRIf slide preparation

Considerations

preparing 4 iRIf PDITC slides for measurement
spot antigens on 2 slides

2015.07.30

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 30 min
slides were incubated in PDITC for 2 h
4 µL of antigens and bBSA were spotted and incubated o/n at 4 °C. For GFP Spot7 was 4 µl and spot8 was 3 µl
in contrast to Experiment 37a the antigens of Tetanus (11) and Salmonella (15) were heatshocked before spotting.

spotting pattern:

#	spot	Elution no.	Concentration	Antibody	AB-conc.[µg/ml]
1-2	HIV(17)	1	1.0 mg/mL??	gp41 DDX1306	20
3-4	HCV(10)	1	0.74 mg/mL??		20
5-6	Tetanus(11)	1	3.75 mg/mL??	HYB 278-01	20
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	Salmonella(15)	2	6.7 mg/mL	a-Salmonella-(pIG15_1301)	100
				anti-His (mouse)	20

2015.07.31

Experiment/protocol

spots were blocked 2x 5 min with PDITC blocking solution
slides were blocked in 10 mg/mL BSA in PBS for 30 min
slides were washed in PBS for 10 min
slides were washed in diluted PBS 2x 10 min
slides were stored in Petri dish at 4 °C (humid atmosphere) until they could be measured in iRIf

Measurement

Flush protocol (Slide 208):

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	45	600	10 mg/ml
Buffer	3	60	600	1x
Anti-GFP(goat;biotinylated)	4	30	600	5 ug/ml
Buffer	5	60	300	1x
Anti-HIV (monoclonal)	6	20	900	20 ug/ml
Buffer	7	60	300	1x
Anti-HCV (monoclonal)	8	20	900	20 ug/ml
Buffer	9	60	300	1x
Anti-Tetanus (monoclonal)	10	30	600	20 ug/ml
Buffer	11	60	300	1x
Anti-HIV (polyclonall)	12	20	900	20 ug/ml
Buffer	13	60	300	1x
Anti-HCV (polyclonal)	14	20	900	20 ug/ml
Buffer	15	60	300	1x
Anti-Salmonella (1301 Elu1)	16	20	900	1:3
Buffer	17	60	300	1x
….further steps were skipped

For second slide flush protocol was changed→ added anti-His step and further dilutions of anti-Salmonella (but threw out a-HIV/a-HCV/anti-Tet) Flush protocol (Slide 12):

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(goat;biotinylated)	4	30	600	5 ug/ml
Buffer	5	60	300	1x
Anti-His	6	30	600	20 ug/ml
Buffer	7	60	300	1x
Anti-Salm (Elu 3)	8	20	900	1:5
Buffer	9	60	300	1x
Anti-Salm (Elu 3)	10	30	600	1:2
Buffer	11	60	300	1x
Anti-Salm (Elu 1)	12	30	600	1:10
Buffer	13	60	300	1x
Anti-Salm (Elu 1)	14	20	900	1:3
Buffer	15	60	300	1x
StrepCy5	16	30	600	5 ug/ml
Buffer	17	60	300	1x

Results

Anti-His at least confirmed that something with a His-Tag is within the HIV and HCV spots (see binding curves of slide 12).

Slide 208: Binding curves; flushing with anti-Salmonella lysate screwed up the measurement → protocol only to this step

Slide 208: Selection of ROIs: As can be seen we faced some problems during that measurement ;)

Slide 12: Binding curves

Slide 12: Selection of ROIs

Experiment 38: Measurement of different GFPs with NanoDrop

2015.07.29

Considerations

determine exact GFP concentration of used stock solution

Experiment/protocol

measured concentration of GFP from different sources via NanoDrop
determined absorption at 488 nm of different GFP solutions and calculated concentration
extinction coefficient EGFP ε = 55000 M^-1cm^-1
layer thickness d= 1 mm
M(GFP)= 26900 g/mol

Results

This measurement:

GFP	concentration [mg/ml]	error [mg/ml]
Max GFP	0.88	0.053 (6%)
Lysate HisGFP	0.81	0.051 (6%)
Lysate noTag GFP	1.25	0.039 (3%)
Weber GFP 8.6	4.05	0.204(5%)
Weber GFP 600	0.38	0.018(5%)

Last measurement from experiment 32 (plate reader):

GFP	concentration [mg/ml]	error [mg/ml]
Lysate HisGFP	0.62	0.17 (27%)
Lysate noTag GFP	1.20	0.31 (26%)
Weber GFP 6.8	7.76	2.04 (26%)
Weber GFP 600	70.60	14.22 (20%)

Experiment 37a: PDITC iRIf slides for measurement with antigens

protocols used :iRIf slide preparation

Considerations

preparing 4 iRIf PDITC slides for measurement
spot freshly purified antigens from ProtPur-group on 2 slides and measure with polyclonal antibodies for HIV and HCV antigen and with monoclonal antibodies for Tetanus and Salmonella (same antibodies that were used in Experiment 36a)

2015.07.29

Experiment/Protocol

prepared 4 iRIf slides with PDITC according to protocol
plasma activation for 5 min at 80 L/h
slides were incubated in APTES solutiion for 1 h 10 min instead of 30 min
slides were incubated in PDITC solution for 2 h 30 min instead of 2 h
3 µL of antigens and controls (Max-GFP and bBSA) were spotted and incubated o/n at 4 °C

spotting pattern:

#	spot	Elution no.	Concentration	Antibody	AB-conc.[µg/ml]
1-2	HIV(17)	2	1.0 mg/mL	gp41 DDX1306	20
3-4	HCV(10)	2	0.74 mg/mL		20
5-6	Tetanus(11)	2	3.75 mg/mL	HYB 278-01	20
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	Salmonella(15)	2	6.7 mg/mL	a-Salmonella-(pIG15_1301)	100
				anti-His (mouse)	20

2015.07.30

Experiment/Protocol

After 23 h of incubation spots were blocked 2x 5 min with PDITC blocking solution
blocked slides 45 min in 10 mg/mL BSA solution
washed 2x in PBS
slide 467 was washed 1x in deluted PBS
slide 215 was washed 2x in deluted PBS
slides were measured in iRIF

Measurement

Step	Solution	Concentration	Amount
1	buffer
2	BSA	10 mg/ml	330 µl
3	buffer
4	anti-GFP	5 mg/mL	300 µL
5	buffer
6	anti-HIV	20 µg/mL	300 µL
7	buffer
8	anti-HCV	20 µg/mL	300 µL
9	buffer
10	anti-Tetanus	20 µg/mL	300 µL
11	buffer
12	anti-Salmonella	1:2	300 µL
13	buffer
14	StrepCy5	5 µg/ml (in PBS:BSA = 1:1)

Resuslts

Experiment was conducted in duplicate. Both measurements showed no antibody/antigen binding. Positive

controls (GFP/anti-GFP, bBSA/Strep) performed well.

Slide 215: Binding curves

Slide 215: ROI selection

Slide 467: Binding curves

Slide 467: ROI selection

Experiment 37b: PDITC iRIf slides for measurement with mouse and rabbit antibodies

protocols used :iRIf slide preparation

Considerations

spot mouse and rabbit antibodies on remaining 2 slides from Experiment 37a and measure with anti-mouse/anti-rabbit

2015.07.29

Experiment/Protocol

3 µL of a antibody out of mouse, the HCV antibody (out of rabbit), GFP-antibody (out of goat - negative control) and positive controls (GFP, bBSA) were spotted on 2 PDITC iRIf slides and incubated o/n at 4 °C

spotting pattern:

#	spot	Concentration	Antibody	AB-conc.[µg/ml]
1-3	mouse AB	1 mg/mL	anti-mouse	20
4-6	rabbit AB (anti-HCV)	1 mg/mL	anti-rabbit	20
7-9	Max-GFP	1.0 mg/mL	a-GFP (mouse;0.5 mg/ml)	5
10	Goat AB (anti-GFP)	0.2 mg/mL	-	-
11	bBSA	0.1 mg/mL	Strep-Cy5	(Strep-Cy5) 10

2015.07.30

Experiment/Protocol

After 23 h of incubation spots were blocked 2x 5 min with PDITC blocking solution
blocked slides 45 min in 10 mg/mL BSA solution
washed 2x in PBS
washed 1x in deluted PBS
slides were measured in iRIF
slide 1: 121 , slide 2: 287

Measurement

Step	Solution	Concentration	Amount
1	buffer
2	BSA	10 mg/ml	470 µl
3	buffer
4	anti-Mouse	20 µg/ml (in PBS:BSA = 1:1)	330 µl
5	buffer
6	anti-Rabbit	20 µg/ml (in PBS:BSA = 1:1)	330 µl
7	buffer
8	anti-GFP	5 µg/ml (in PBS:BSA = 1:1)	330 µl
9	buffer
10	StrepCy5	5 µg/ml (in PBS:BSA = 1:1)	330 µl
11	buffer

Results

The secondary antibodies bound properly to their corresponding antibodies derived from mouse/rabbit.

Slide 121: binding curves

Slide 121: ROI selection

Slide 287: binding curves - different a-GFP was tested which did not bind to GFP

Slide 287: ROI selection

Experiment 36b: Ni-NTA slides for iRIf group - cell free expressed tYFP

2015.07.27

Considerations

Spot cell free expressed YFP and mCherry-Lysate on remaining 2 iRIf slides from experiment 36a

Experiment/Protocol

3 µL of cell free expressed protein, mCherry and controls were spotted on the Ni-NTA slides from 2015.07.23. and incubated o/n

spotting pattern:

#	spot
1	pIG15-104 Bernhard
2	pIG15-105 Bernhard
3	mCherry Lysate (HA-Tag)
4	pIG15-104 Promega
5	pIG15-105 Promega
6	negative control with Promega
10	pIG15-104 Retikulo
11	pIG15-105 Retikulo
7+8	Max-GFP 1mg/mL
9	bBSA 100µg/mL

protein spots incubated for 24 h at 4 °C

2015.07.28

Experiment/Protocol

Spots were blocked 2x 5 min with BSA 10 mg/ml at room temperature
Slides were flushed with PBS 1x and spotting mask was removed
Washing steps in slideholder at 4°C followed: 10 min PBS 1x, 10 min PBS + 20 mM Imidazol and 10 min 1/10 diluted PBS (in aqua dest)
Then blocking for 30 min in BSA 10 mg/ml at 4°C followed
Slides were washed for 10 min in PBS 1x and 10 min in 1/10 diluted PBS (in aqua dest) at 4°C
After drying with wafer gun, fluorescence of YFP was checked in Microarray Scanner
Slides were then measured in iRIf

Measurement

Slide 1

Step	Solution	Concentration	Amount
1	buffer
2	BSA	10 mg/ml	330 µl
3	buffer
4	anti tYFP	20 µg/ml (in PBS)	340 µl
5	buffer
6	anti-His	10 µg/ml (in PBS)
7	buffer
8	anti-GFP (goat, biotinylated)(in PBS:BSA = 1:1)	5 µg/ml
9	buffer
10	anti-HA	??? von Norman, 3 weeks old
11	buffer
12	StrepCy5	5 µg/ml (in PBS:BSA = 1:1)

Slide 2

Step	Solution	Concentration	Amount
1	buffer
2	BSA	10 mg/ml	330 µl
3	buffer
4	anti tYFP	ca. 17 µg/ml (in PBS)	350 µl
5	buffer
6	anti-GFP (goat, biotinylated)(in PBS:BSA = 1:1)	5 µg/ml	340 µl
7	buffer
8	StrepCy5	5 µg/ml (in PBS:BSA = 1:1)	330 µl

Results

there might be a very weak a-tYFP/tYFP binding
concentration of expressed tYFP is too low for a convincing signal

Binding curve for S1: Only with lots of zooming anti-tYFP can be seen to bind tYFP (spot 4) within the binding curves (Exp. 36b)

Quotient picture (before/after anti-tYFP): Weak signal for Spot 4 can be seen

Binding curve for S2: Only with lots of zooming anti-tYFP can be seen to bind tYFP (spot 4) within the binding curves (Exp. 36b)

Quotient picture that was taken for spot marking

Quotient picture (before/after anti-tYFP): Weak signal for Spot 4 can be seen

Experiment 36a: Ni-NTA slides for iRIf group - antigens

protocols used :Plasma activation, APTES + PDITC surface, Ni-NTA

2015.07.22

Considerations

prepare Ni-NTA slides for the iRIf group to check the antibody binding to our expressed antigens

Experiment/Protocol

prepared 4 iRIf PDITC slides according to protocol
plasma activation time: 5 min
sandwiched them with NTA and incubated o/n at 4°C

2015.07.23

Experiment/Protocol

slides were blocked in APTES blocking solution for 1h in slideholder
washed 2x 10 min with PBS (shaky, shaky)
incubated slides in freshly prepared 1% NiSO₄ solution for 1h
washed slides 2x 10 min in PBS and then 1x in 1/10 diluted PBS (shaky, shaky)
spotted 3 µl antigen samples and controls (Max GFP, bBSA)and incubated them overnight

spotting pattern:

#	spot	Elution no.	Concentration	Antibody	AB-conc.
1-2	HIV(17)	2	0.36 mg/ml	gp41 DDX1306	20 ug/ml
3-4	HCV(10)	Mix (1-3)	0.13 mg/ml		20 ug/ml
5-6	Tetanus(11)	Mix (1-3)	0.55 mg/ml		20 ug/ml
7-8	GFP		1.0 mg/ml	a-GFP (goat)	5 ug/ml
9	bBSA		0.2 mg/ml	Strep-Cy5	10 ug/ml
10-11	Salmonella(15)	2	3.2 mg/ml	a-Salmonella-Lysate (13)	400 ug/ml (lysate conc.)

803 - (0.13 mg/ml) - not spotted
Two slides were spotted. For first slide the (max.) concentrations of the Antigens were used. For the second slide the second spot of each Antigen was spotted diluted (100 ug/ml).

2015.07.24

Experiment/Protocol

After incubating for 13h at 4°C all spots were removed with pipette
slides were then blocked with BSA (10 mg/ml) 2x 10 min at room temperature
1x PBS buffer was flooded over slides before removing the spotting mask and washing in PBS 2x 10 min (each time stored at 4°C)
Then a washing step in 1/10 diluted PBS (with aqua dest.) followed (also at 4°C)
Slides were dried with wafer gun and stored at 4°C in humid atmosphere
After 15 min slides were put back in PBS for 10 min because of Stefan
Talking to Günter confirmed that storing the slides dry prevents the His-Tag from leaving Ni/NTA so the slides were dipped into PBS (1/10 diluted, dried with wafer gun and stored at 4°C in humid atmosphere
slides were blocked again in BSA 10 mg/ml for 30 min
Then they were washed 10 min in PBS 1x and 10 min in 1/10 diluted PBS (in aqua dest), eacht time at 4°C
Slides were dried and measured in iRIf

Measurement

Flush protocol for both slides

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	60	500	10 mg/mL
Buffer	3	60	510	1x
Anti-GFP(goat;biotinylated)	4	20	920	5 ug/mL
Buffer	5	60	300	1x
Anti-HIV (polyclonal)	6	20	900	20 ug/mL
Buffer	7	60	300	1x
Anti-HCV (polyclonal)	8	20	900	20 ug/mL
Buffer	9	60	300	1x
Anti-Tetanus (mk: HYB 278-15)	10	20	900	20 ug/mL
Buffer	11	60	300	1x
Strep-Cy5	12	30	600	5 ug/mL
Buffer	13	60	300	1x
Anti-His	14	30	600	20 µg/mL
Buffer	15	60	300	1x
Anti-Salmonella (1301 Elu2)	16	20	900	1.6 mg/mL 1:4
Buffer	17	60	300	1x

Results

No specific antigen/antibody binding
positive control (GFP) worked
negative control didn't work (bBSA was detectable with Strep-Cy5 although it should not bind to Ni-NTA)
Anti-His at least bound to Tetanus and Samonella
monoclonal antibodies did'nt bind
for HIV, HCV: maybe concentration was too low

Binding curve of the second slide: No specific antibody/antigen binding can be seen

Binding curve of S1: results are equal to S2 (anti-Salm not present within this plot, since huge airbubble before the step destroyed further measurement)

Experiment 35: Influence of focus on fluorescence intensity

2015.07.21

Considerations

check if there is an impact on the fluorescence intensity when the photo is taken out of focus (compared to in focus photo)

Experiment/Protocol

three photos of a spot were taken and evaluated, one of them in focus, the others out of focus

Results

Compared fluorescence intensities of images from the same spot taken with different focus calibrations

there is no dependency between the fluorescence intensity and the focusing

Experiment 34: Influence of objcetive on fluorescence intensity

2015.07.21

Considerations

check if fluorescence images made with the 10x objective are brighter than with the 4x objective
how much brighter

Experiment/Protocol

made image of the same spot with 10x objective as well as the 4x objective
measured 3 spots and compared fluorescence intensities

Results

Compared fluorescence intensities of images from the same spot taken with the 10x objective and the 4x objective

as expected the images taken with the 10xobjective are brigther
3-10 time as bright as the 4x objective images
comparison of images taken with the 4x and 10x objectives is difficult → use only one objective for spot evaluation

Experiment 33: Photobleaching of GFP

2015.07.21

Considerations

determine impact of photobleaching on the fluorescence measurement of GFP

Experiment/Protocol

took a photo every minute of 3 individual spots for 10 min while constantly illuminating them with blue light
slides from experiment 30 were used

Results

Impact of photobleaching of GFP spots on measured fluorescence intensities.

→ photobleaching effect negligible for the duration of our experiments

Experiment 32: Dilution series of GFP III and fluorescence measurement

2015.07.20

Considerations

repeat dilution series with different range of dilutions since no linearity could be observed in the previous experiment 29

Experiment/Protocol

Scheme for dilutions in plate reader:

	1	2	3	4	5	6	7	8	9	10
A	PBS	PBS	PBS
B Max GFP	pure	1:2	1:4	1:8	1:10	1:15	1:20	1:30
C Max GFP	pure	1:2	1:4	1:8	1:10	1:15	1:20	1:30
D Max GFP	pure	1:2	1:4	1:8	1:10	1:15	1:20	1:30
E Lysate His-GFP	1:2	1:2	1:5	1:5	1:10	1:10	1:20	1:20	1:40	1:40
F Lysate GFP no Tag	1:2	1:2	1:5	1:5	1:10	1:10	1:20	1:20	1:40	1:40
G Weber GFP 8.6 mg/ml	1:20	1:20	1:50	1:50	1:100	1:100	1:200	1:200	1:500	1:500
H Weber GFP 600 mg/ml	1:200	1:200	1:500	1:500	1:1000	1:1000	1:2000	1:2000	1:4000	1:4000

Results

First Measurement

Second Measurement with shaking (30s)

2015.07.21

Evaluation

Measured fluorescence values were corrected by mean intensity for PBS
for each dilution the mean value of the corrected intensities was calculated
A calibration curve was made from the values for Max GFP (c = 1mg/ml)

The concentrations of the other GFP solutions were calculated by using the equation obtained from the calibration and multiplying with the dilution factor.
The final concentration was obtained by calculating the mean value for all 5 dilutions. The error corresponds to the standard deviation of these 5 values.

GFP	concentration [mg/ml]	error [mg/ml]
Lysate HisGFP	0.62	0.17 (27%)
Lysate noTag GFP	1.20	0.31 (26%)
Weber GFP 6.8	7.76	2.04 (26%)
Weber GFP 600	70.60	14.22 (20%)

Experiment 31: Ni/NTA slides for iRIf

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface, Ni-NTA

2015.07.19

Experiment/Protocol

2 iRIf slides were made until after loading the NTA surface with Ni
slides were plasma activated for 5 min at gas flow 80 L/h
Spotting pattern:

#	spot
1-3	APTES+PDITC+NTA+Ni+Lysate His-GFP
4-6	APTES+PDITC+Lysate His-GFP
7-9	APTES+PDITC+NTA+Ni+Max GFP
10	APTES+PDITC+NTA+bBSA
11	APTES+PDITC+bBSA

slides were incubated in PDITC solution for 3h15min
NTA solution from 04.07.2015 was used
spotted slides were incubated overnight in the fridge at 4°C and humid atmosphere

2015.07.20

Experiment/Protocol

slides were blocked by spotting APTES blocking solution on all spots (also PDITC-spots).
slides were washed 2x in PBS in petri dish
NiSO₄ was spotted on the NTA-spots and incubated for 1h15min
Slides were washed 2x with PBS + 1x with diluted PBS in glass vase
GFPs were spotted according to spotting pattern and incubated o/n at 4°C

2015.07.21

Experiment/Protocol

GFP was removed from slide and spots were incubated 2x with BSA (10 mg/mL)for 5 min
slides were floated with PBS, then washed 2x with PBS in slide holder for 10 min
slides were washed with deluted PBS (1:10) + Imidazole (20 mM)
slide 1 was again washed in deluted PBS (1:10) and then measured in iRIf
slide 2 was stored in deluted PBS (1:10)

Measurement

Step	Solution	Concentration	Amount
1	buffer
2	BSA	10 mg/ml	330 µl
3	buffer
4	anti-GFP (goat, biotinylated)(in PBS:BSA = 1:1)	5 µg/ml	340 µl
5	buffer
6	StrepCy5	5 µg/ml (in PBS:BSA = 1:1)	330 µl

Results

slide 1

slide 2

slide 1 was full of salt, therefore slide 2 was washed longer in deluted PBS
for slide 2 the blocking step wasn't long enough, we should block more

Experiment 30: SaNSibar 2.0

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface, Ni-NTA

2015.07.19

Experiment/Protocol

replicate experiment 27 from 30.6.2015
Each slide was treated only by one person.
Spotting pattern:

#	spot
1-3	HisGFP lysate	on Ni/NTA
4-6	GFP no tag lysate
7-9	Max GFP
10-12	Weber GFP 600 mg/ml
13-15	HisGFP lysate	only on APTES/PDITC, no NTA
16-18	GFP no tag lysate
19-21	Max GFP
22-24	Weber GFP 600 mg/ml
25	bBSA	on Ni/NTA
26	bBSA	only on APTES/PDITC, no NTA

slides were incubated in PDITC solution for 3h15min
NTA solution from 04.07.2015 was used
spotted slides were incubated overnight in the fridge at 4°C and humid atmosphere

2015.07.20

Experiment/Protocol

slides were blocked by spotting APTES blocking solution on the NTA-spots for 1 hour
on Sa-slide the blocking solution was also spotted on the PDITC-spots
slides were washed 2x in PBS in petri dish
NiSO4 was spotted on the NTA-spots and incubated for 1h15min
Slides were washed 2x with PBS + 1x with diluted PBS in glass vase
GFPs were spotted according to spotting pattern and incubated o/n at 4°C
on Sa-slide there is 1 additional His-GFP-Lysate spot below spot nr. 15

2015.07.21

Experiment/Protocol

GFP was removed from slide and spots were incubated 2x with BSA (10 mg/mL)for 5 min
slides were floated with PBS, then washed 2x with PBS in slide holder for 10 min
slides were washed with deluted PBS (1:10) + Imidazole (20 mM)
GFP fluorescence was measured
slides were incubated with Strep-Cy5 for 45 min and washed according to protocol
Cy5 fluorescence was measured

2015.07.21

Results

Mean GFP Fluorescence Intensity of all slides + Mean Background for each slide.

Mean Cy5 Fluorescence Intensity of all slides + Mean Background for each slide

on Si slide the second bBSA spot (on PDITC) is missing (wasn't spotted)

Experiment 29: Measured fluorescence of dilution series of GFP to determine concentration

2015.07.18

Scheme for dilutions in plate reader:

	1	2	3	4	5	6	7	8	9	10	11
A	H₂O	Max GFP 1:10	Max GFP 1:20	Max GFP 1:30	Max GFP 1:40	Max GFP 1:50	Max GFP 1:60	Max GFP 1:70	Max GFP 1:80	Max GFP 1:90	Max GFP 1:100
B	PBS	Lysate His-GFP 1:10	Lysate His-GFP 1:20	Lysate His-GFP 1:40	Lysate His-GFP 1:80	Lysate His-GFP 1:100
C		Lysate GFP no Tag 1:10	Lysate GFP no Tag 1:20	Lysate GFP no Tag 1:40	Lysate GFP no Tag 1:80	Lysate GFP no Tag 1:100
D		Weber GFP 8.6 mg/ml 1:50	Weber GFP 8.6 mg/ml 1:100	Weber GFP 8.6 mg/ml 1:200	Weber GFP 8.6 mg/ml 1:500	Weber GFP 8.6 mg/ml 1:1000
E		Weber GFP 600 mg/ml 1:100	Weber GFP 600 mg/ml 1:500	Weber GFP 600 mg/ml 1:700	Weber GFP 600 mg/ml 1:1000	Weber GFP 600 mg/ml 1:2000

Results

No linear correlation was obtained for the Max GFP dilutions.
The fluorescence intensity of the majority of the chosen Max GFP dilutions was very low.
–> Experiment should be repeated with different dilutions (less diluted) and triplicates in order to obtain a calibration curve.

Experiment 28: Comparison of different GFPs on old Ni/NTA surfaces from 2015.06.15

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface, Ni-NTA

2015.07.06

2 GOPTS and 2 APTES/PDITC slides, each modified with Ni/NTA from 15.06.2015, Experiment 18 (Had been stored in the fridge at 4°C since then)
For spotting 11-well spotting mask was used. Each well was filled with 3 µl solution.
Spotting pattern:

#	spot
1-3	HisGFP lysate
4-6	GFP lysate no Tag
7-9	Weber GFP 8.6 mg/mL / For 2 slides 600 mg/mL (marked with black points in upper left-hand corner)
10	bBSA
11	bBSA

2015.07.07.

protein solutions were removed with pipette
spots were blocked with 10 mg/ml BSA solution for 5 min
spotting masks were removed
slides were washed 2×10 min in PBS buffer

Results

Mean Fluorescence Intensity of different GFPs on old NTA slides_all slides each spot.

Mean Fluorescence Intensity of different GFPs on old NTA slides_only PDITC slides Mean of 3 spots per GFP.

Mean Fluorescence Intensity of bBSA-Strep-Cy5 on old NTA slides.

Experiment 27: Comparison of different GFPs on Ni/NTA APTES/PDITC surface, replicate 1 (SaNSibar)

protocols used :Plasma activation, APTES + PDITC surface, Ni-NTA

2015.07.06

3 slides were prepared according to the protocols.
Each slide was treated only by one person.
Spotting pattern:

#	spot
1-3	HisGFP lysate	on Ni/NTA
4-6	GFP no tag lysate
7-9	Max GFP
10-12	Weber GFP 8.6 mg/ml
13-15	HisGFP lysate	only on APTES/PDITC, no NTA
16-18	GFP no tag lysate
19-21	Max GFP
22-24	Weber GFP 8.6 mg/ml
25	bBSA	on Ni/NTA
26	bBSA	only on APTES/PDITC, no NTA

spotted slides were incubated overnight in the fridge at 4°C and humid atmosphere

2015.07.07

protein solutions were removed with pipette
slides were blocked with BSA 5 mg/ml + 5% ethanolamine in PBS for 45 min
slides were washed 2×10 min in PBS buffer without removing spotting mask

Results

Mean GFP Fluorescence Intensity out of three spots + mean background over whole slides.

Mean Cy5 Fluorescence Intensity + mean background.

–> His-GFP-Lysate binds best on Ni-NTA, Weber GFP binds best on PDITC.

Experiment 26: Spotted NTA on PDITC slides to determine amount of unspecific binding

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface, Ni-NTA

2015.07.02

considerations

determine unspecific binding on Ni-NTA surface

Experiment/Protocols

prepared 3 APTES/PDITC slides according to APTES + PDITC surface
spotted 2 µl NTA (457 mM in 1 M NaHCO₃)using the spotting templates
spotting pattern:

#	spot
1-3	PDITC + NTA + Blocking + His-GFP lysate
4-6	PDITC + NTA + Blocking + Weber His-GFP 8 mg/mL
7-8	PDITC + NTA + Blocking + nT-GFP lysate
9	PDITC + BSA
10	PDITC + Spotting control (cy5 labeled DNA)
11	PDITC + positive control (nT-GFP lysate)

2015.07.03

Experiment/Protocol

slides were washed with PBS according to Ni-NTA with spotting mask still on slide
1 % Nickelsulfate solution was spotted on top of the NTA spots (Spot 1-8)
slides were again washed with PBS according to Ni-NTA with spotting mask still on slide
GFP, His-GFP, BSA and Cy5-labeled DNA were spotted as mentioned in the table above (Experiment 25, 2.7.2015)

2015.07.04

Experiment/Protocol

removed protein and DNA solution with pipette
incubated twice with 4 µl of BSA (10mg/ml) for 5 min
washed twice with PBS for 10 min
washed with 10% PBS with 20mM imidazole (27,2 mg in 20 ml PBS) for 10 min
washed with 10% PBS for 10 min
dried slides with nitrogengun and measured GFP fluorescence

Results

in the diagrams the results for slide 2 and 3 are shown. On slide 1 not all spots were visible.

Mean Fluorescence Intensities of slides 2 and 3 with measured background for each spot.

Mean Fluorescence Intensities of slides 2 and 3 with background already substracted.

The diagramm shows, that His-GFP was successfully immobilized on the Ni-NTA surface, while GFP without Tag didn't bind very good
the fluorescence of our lysate was much stronger than that of the purified His-GFP
the positive control (nT-GFP on PDITC) didn't show intense fluorescence, because nT-GFP-Lysate was used

To-Do

Repeat dilution series (2 slides, 5 concentrations: 2 spots each, 1:1, 1:4, 1:16, 1:64, 1:256)
shutterspeed linearity –> ask Max
re-evaluate shutterspeed linearity with NEF-Data from the old dataset (26.05.15)
Talk to Günther
- about what to do next
- reference values for their Cy5 (How good are our slides?)
- GFP - why no homogenous spots but rings
- test if His binds to Ni or to PDITC/NTA by making NTA-spots without Ni

Store slides for GFP incubation in fridge in plastic dishes with top of the dish beneath the slide (dish upside down)

spin GFP before use to separate conglomerates from solution

Experiment 25: Check detectibility of GFP bound on PDITC slides in the iRIf setup

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface

2015.07.01

Considerations

repeat experiment from 2015.06.30 (different GFPs, mCherry, bBSA, bDNA, Cy5DNA on PDITC) on 2 iRIf slides

New 2 ml Eppi of fresh APTES from abcr was prepared and used

Experiment/Protocols

3 iRIf slides
surface was prepared according to Plasma activation and APTES + PDITC surface
incubation in PDITC solution was 5h instead of 2h
slides were spotted (2 µl spots) according to the following pattern:

#	spot
1	Lysate GFP no tag
2	Lysate GFP-2x6His
3	Max' GFP-His
4	Weber GFP 8.6 mg/mL
5	Weber GFP 600 mg/mL
6	mCherry
7	BSA 10 mg/mL
8+9	bBSA 50 ng/µL
10	spotting control 2 µL
11	binding control 0.5 µL

Missing corner of spotting mask was in the upper left hand corner of the slide (slide 1: number on left side,

Results

due to defects of the iRIf setup no measurement was taken
the iRIf-slides were cleaned for reuse

Experiment 24: Compare quality of own PDITC surface with that of AG Roth

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface

2015.07.01

Considerations

compare our slides with slides from Norman

New 2 ml Eppi of fresh APTES from abcr was prepared and used

Experiment/Protocol

one of Norman's slides prepared by Norman and one of our slides prepared by us
2 of the prepared iRIf-PDITC-slides were spotted according to the following pattern/table
GFP was taken away from well with pipette and immediately replaced by 10 µl of BSA (10 mg/ml)
this step was carried out 2x
slides were then washed with PBS from squeeze bottle, the spotting mask was removed and the slides were immediately put into a slideholder filled with PBS/BSA for 15 min
GFP stock concentrations with 0.5 mg/mL
on our slide the BSA spot is left to the bBSA spot
on Normans slide the BSA spot is right to the bBSA Spot

#	spot
1	GFP Norman 1:10
2	GFP Norman 1:30
3	GFP Norman 1:100
4	GFP Max 1:10
5	GFP Max 1:30
6	GFP Max 1:100
7	GFP Weber 1:10
8	GFP Weber 1:30
9	GFP Weber 1:100
10	bBSA 0.5 mg/mL, 50 %
11	BSA 10 mg/mL

2015.07.02

Experiment/Protocol

used slides prepared and spotted on 1.7.2015
GFP was taken away from well with pipette and immediately replaced by 10 µl of BSA (10 mg/ml)
this step was carried out 2x
slides were then washed with PBS from squeeze bottle, the spotting mask was removed and the slides were immediately put into a slideholder filled with 5mg/ml BSA/PBS for ca. 15 min
washed slides with aqua dest before putting it into the iRIf setup
iRIf programm:
- blocking with 330 µl BSA (5mg/ml in PBS)
- flushing with 330 µl anti-gfp (5 µg/ml in 5 mg/ml BSA in PBS)
- flushing with 330 µl streptavidin-cy5 (5 µg/ml in 5 mg/ml BSA in PBS)

Results

blocking step iGEM slide

Anti-GFP binding iGEM slide

Strep-Cy5 binding iGEM slide

Strep-Cy5 Fluorescence 100% Laser iGEM slide

Strep-Cy5 Fluorescence 100% Laser Norman slide

Experiment 23: Second Replicate: Comparison between different GFPs (Own,Max,Weber), replicate 2 of experiment 22

protocols used :Plasma activation, GOPTS surface, APTES + PDITC surface

2015.07.02

Considerations

check reproducibility of strong fluorescence intensities from experiment 21

Experiment/Protocols

made 4 GOPTS and 4 APTES/PDITC slides in accordance with the protocols (GOPTS surface, APTES + PDITC surface)
repeated experimental procedure from experiment 21

2015.07.03

Experiment/Protocols

the o/n with GFP, mCherry, etc.incubated slides were used
GFP was taken away from well with pipette and immediately replaced by 10 µl of BSA (10 mg/ml)
this step was carried out 2x
slides were then washed with PBS from squeeze bottle, the spotting mask was removed and the slides were immediately put into a slideholder filled with PBS/BSA for 15 min
fluorescence intensity was measured

Results

Evaluation of GOPTS slides

Evaluation of PDITC slides

Team:Freiburg/Labjournals/SurChem/August

Labjournal Surchem July

Experiment 39b: PDITC iRIf slides for measurement with PhyA

Considerations

2015.07.30

Experiment/protocol

2015.07.31

Experiment/protocol

2015.08.02

2015.08.03

Experiment/Protocol

Measurement

Slide 1

slide 2

Results

Experiment 39a: PDITC iRIf slides for measurement with purified antigens

Considerations

2015.07.30

Experiment/protocol

2015.07.31

Experiment/protocol

Measurement

Results

Experiment 38: Measurement of different GFPs with NanoDrop

2015.07.29

Considerations

Experiment/protocol

Results

Experiment 37a: PDITC iRIf slides for measurement with antigens

Considerations

2015.07.29

Experiment/Protocol

2015.07.30

Experiment/Protocol

Measurement

Resuslts

Experiment 37b: PDITC iRIf slides for measurement with mouse and rabbit antibodies

Considerations

2015.07.29

Experiment/Protocol

2015.07.30

Experiment/Protocol

Measurement

Results

Experiment 36b: Ni-NTA slides for iRIf group - cell free expressed tYFP

2015.07.27

Considerations

Experiment/Protocol

2015.07.28

Experiment/Protocol

Measurement

Results

Experiment 36a: Ni-NTA slides for iRIf group - antigens

2015.07.22

Considerations

Experiment/Protocol

2015.07.23

Experiment/Protocol

2015.07.24

Experiment/Protocol

Measurement

Results

Experiment 35: Influence of focus on fluorescence intensity

2015.07.21

Considerations

Experiment/Protocol

Results

Experiment 34: Influence of objcetive on fluorescence intensity

2015.07.21

Considerations

Experiment/Protocol

Results

Experiment 33: Photobleaching of GFP

2015.07.21

Considerations

Experiment/Protocol

Results

Experiment 32: Dilution series of GFP III and fluorescence measurement

2015.07.20

Considerations