5 Slides Ni-NTA (3X Serum(-)/Serum(+), 2x Serum (-)/a-GFP)

2 Slides PDITC (2x Serum(-)/Serum(+))

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

Spotting (Slide 303):

Spotting (Slide 429):

Spotting:

Flush protocol:

14.08.2015

Spotting:

Flush protocol:

Roi selection Exp. 46b: Spot 11 couldnt be selected due to air bubble on it

Binding curves of Exp. 46b for all spots

Binding curves of Exp. 46b only for spots 1-6

Spot 11 couldnt be evaluated due to air on it. Spot 1-3 show a-HA binding (weak but clear) and good a-GFP binding → successful cellfree expression No strepcy5 binding can be detected: Tube to flowchamber pulled off just before that step….

13.08.2015

spotting pattern:

Dilute antigens in TBS (if necessary)!

Dilute antibodies in TBS –> BSA in TBS

2 replicates on PDITC: (024 & 502)

2 Replicates on NiNTA (501&502)

2 Replicates on NiNTA (216 & 476)

Flush protocol (for Ni-NTA slides):

one Ni-NTA slide 1 anti-mouse was changed against anti-his!!! one Ni-NTA slide could not be measured due to hydrophobicity problems

Flush protocol (for PDITC slide 502):

No antigen/antibody binding (HIV/HCV) was detected on the Ni-NTA slides. Controls worked fine.

Flush protocol (for PDITC slide 24):

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)

11.08.2015

spotting pattern:

Slide 254: Roi selection

Slide 254: Binding curves. Binding curve shows (better to see with zoom) little a-tYFP (from rabbit) binding to Kokos tYFP spot (Mg2+ fed). Signal is amplified with a-Rabbit

Slide 254: Quotient picture (before/after a-tYFP step)

Slide 254: Quotient picture (before/after a-Rabbit step)

11.08.2015

Spotting:

Duplicates were prepared (Slide 12 and slide 121) but only 1 could be measured, because slide 121 broke ( 8[ ) → Only slide 12 was evaluated.

Slide 12: Selection of ROIs

Slide 12: Binding curves of all spots

Slide 12: Binding curves of PhyA spots

Slide 12: Binding curves of Max GFP spots

08.08.2015 Spotting:

All spots diluted to proteinconcentration of ~0.4-0.5 mg/ml. Antigens were spinned before spotted.

Flush protocol:

Again no antibody-binding was detected → We confirmed that in this manner the experiment does not work Binding curves were evaluated, but revealed no further information. Due to the large protocol and some (air)problems (see binding curves of slide 465) during measurement in both cases, curves do not look neat.

Slide 265: Binding curves; some spots were expluded from the evaluation due to air that came on top of them during measurement

Slide 465: Binding curves; this is what it looks like if you do not exclude the spots were air arrived during measurement (spot 2&8) from the evaluation

06.08.2015

spotting pattern:

Slide 426 - measured in old setup

• slide wasn't blocked before measurement

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

Slide 423 - measured in new setup

Slide 466 - measured in old setup

For evaluation of Halo-Experiment → look up in labjornal of the surface chemistry

04.08.2015

spotting pattern:

Only 1 slide was measured. Second slide syringe didnt suck solutions. Results: Antigens didnt bind to the immobilized antibodies. Strangely, 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

31.07.2015

In contrast to the previous experiments 15 (Salmonella) and 11 (Tetanus) were heatshocked just before spotting (to denature the antigens → maybe AB binds better to the denatured antigens (the primary structure)!?)

Flush protocol (Slide 208):

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):

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

03.08.2015 Flush protocol:

Results: Binding of anti-phyA to phyA was not observed in RIfs. Yet, at both measurements was faced some problems: much of the protein was washed away from the spotted spot. → We have to take lower concentrations for spotting (100-400 ug/ml) Anti-tYFP to YFP binding could not be detected aswell: Actually, this wont work on PDITC since all of the expression mix proteins also bind to the spot, compete with the (already low concentration) YFP for binding → almost no YFP can bind to the spot. We have to repeat experiment for cellfree expressed YFP/GFP with specific surface (e.g. Ni-NTA)

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

30.07.2015

Spotted:

Flush protocol:

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

30.07.2015

Spotted:

Flush protocol:

Results: 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

28.07.2015

Spotting pattern:

Results: They may be (proabaly) a very weak a-tYFP/tYFP binding. Yet, the concentration of cell free expressed tYFP is probably too less to see a convincing signal.

* protein spots incubated for 24 h at 4 °C

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

24.07.2015

Spotting pattern:

pIG_803 / Rubella - (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 µg/ml). Incubation o/n for the binding to the surface.

After taking of the spotting mask and washing with PBS, slides were kept in fridge (4°) under wet conditions.

Measuring: No specific antigen/antibody binding could be detected (see graph of the binding curves). Anti-GFP signal (positive control) was good. Strep-Cy5 signal (which bound to the biotinylated a-GFP aswell) was good. Yet, the presents of the (not His-tagged) bBSA –> unspecific binding. Anti-His binding revealed that at least Tetanus and Salmonella antigen (or something with a His-Tag) are present on the slide. Unfortunately, the monoclonal antibodies do not bind to the expressed and spotted antigens. In case of HIV and HCV also the concentration of the antigen might be limiting.

Binding curve of S2: 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)

20.07.2015 The antigens were stamped with PDMS stamps (50µl each) on slides (S8,S9) (see sceme of the setup). Blocking was performed for 30 min with 2 mg BSA and by contact with a PDMS slide. For each antigen, 330 µl of antibody solution was run over the slide during the measurement. To assure good binding, an excess of antibody was used (20 µg/ml instead of the normally sufficient 5 µg/ml)

Results (S9): GFP/anti-GFP binding was observed, however none of the antigen/antibody pairs showed any binding. Since anti-GFP bound successfully to GFP, mistakes in slide preparation can be excluded, hinting that the antigen/antibody couples are either not able to bind due to wrong epitopes, or that too many proteins are co-purified with the antigens, leading to a very low antigen concentration on the stamps. Westerblots would be an advisable next step to assure that the antibodies are capable of binding to the epitopes of the corresponding antigens.

The anti-salmonella (pIG15_1303) showed strong unspecific binding on the whole slide, confirming previous observations. This might still be due to denaturation problems with the salmonella elutions.

Sceme of the setup

GFP Positive control - Binding of anti-GFP to GFP is visible

21.07.2015 For the duplicate slide (S8), which was prepared in the same manner, even higher antibody concentrations were used to exclude concentration limitations.

Again, a-GFP to GFP binding could be detected well. Unfortunately, no binding of any antibodies to the antigens could be observed. Binding curve analyses follows…

Binding curves slide S8: Evaluation of the binding curves confirms that no binding process for any antibody/antigen pair occured

19.07.2015 A stamp with freshly, cell-free expressed YFP (approx 20µl) was stamped onto an iGEM stamp. A GFP stamp was stamped as control. Blocking was performed for 30min with 10mg/ml BSA, then blocked with a PDMS slide. GFP/Anti-GFP Binding could be observed in iRIF, however no binding of anti-YFP, nor anti-HIS could be detected on the YFP stamp.

19.07.2015 Stamped freshly eluted Salmonella antigen, which was prepared the same day. Apart from not being frozen, salmonella antigen and antibody we're both eluted in a phosphate free buffer. While stamping, it was observed that the salmonella antigen (1501) already showed some flakes floating in the solution, indicating that the antigen is either denaturated, or that it naturally forms aggregates.

The Salmonella Antibody was diluted 1:10. Antigen was stamped without dilution (~3,2 mg/ml protein in solution). Controls were GFP and bBSA stamps. Binding of Salmonella antigen to salmonella antibody could not be observed.

07.07.2015

Salmonella-antigen (1501) and Salmonella-antibody (1301) were provided by the protein purification. Unfortunately, after thawing antibody and antigen, both precipitated. Nevertheless, we continued (with not too great expectations).

To test salmonella-antibody binding to antigen the following experiment was conducted: Stamping sceme (look setup picture). Flush protocol: - PBS (baseline) - BSA (blocking) (500 ug/ml) - PBS - Anti-Salmonella (1301) (0.5 mg/ml!) - PBS - Anti-GFP (5 ug/ml) - PBS - bBSA (200 ug/ml)

Some airbubbles came into chamber by initial flooding. Continued measurement. Blocking was fine. When anti-Salmonella was flushed over the slide, an intensense signal on the whole slide was seen; this is probably due to high unspecific binding. Then (as can be seen within the graph of the binding curves) specific binding of the a-Salmonella to the Salmonella-antigen stamp is detectable. Positive control (GFP / anti-GFP) was positive. bBSA to StrepCy5 was not detectable - this was not that surprising; already plate reader after stamping process revealed low transfer of StrepCy5 to the surface / denaturation of the protein. –> We have to repeat the experiment with unprecipitated antibody/antigen, maybe another surface for less unspecific binding and longer baseline/blocking/binding steps, but in general the antibody seems to bind to the antigen (see increase in intensity within a-Salmonella-step at the Salmonella spots - delta to the baseline)

Setup of the experiment

Evaluation of binding experiment with 1501/1301

07.07.2015

Investigating optimal stamping technique:

Pressure (slide 467): 3 stamps StrepCy5 (50 ug/ml) → without pressure (just put on the slide)/pushed very soft/pushed hard - all incubated for ~30s

Incubation time (Slide 303): 3 stamps StrepCy5 (50 ug/ml) → 1 minute / 5 minutes / 30 minutes - all stamps without pressure As can be seen in the platereader pictures, no differences between different pressures or incubationtimes is detectable.

Stamping with different pressures (constant incubation ~1min): Top → no pressure, bottom right → minimum pressure, bottom left → high pressure

Stamping with different incubation time (constant pressure (no pressure): Top → 1 minute, bottom right → 5 minutes. bottom left → 30 minutes

01.07.2015 This experiment focuses on trying to see whether our salmonella antigen (pIG15_1501) is bound by our salmonella antibody (pIG15_1301). pIG15_1501 (Salmonella antigen) eluation 1 was taken from the -80 freezer. When melting the ~10mg/ml tube, we observed that the antigen appeared to be denaturated, hinting that the storage conditions were not tolerated by the protein. The supernatant was used nonetheless, hoping that some protein might still be correctly folded. The antibody (0,5 mg/ml) was diluted 1:2 in PBS. A plasma-activated slide was used for measurement. A DiaCHIP stamp was incubated with undiluted antigen supernatant. An iGEM stamp was incubated with StrepCY5, an antibody-shaped stamp was incubated with GFP (200µg/ml) for 30 min. After blocking the slide in BSA, a fluorescence measurement was performed to control for correct stamping. The StrepCY5 stamp showed hardly any fluorescence in the plate reader. This indicates bad stamping (wrong pressure, hardly any pressure was applied this time) or faulty StrepCY5.

Anti-Salmonella showed stong unspecific binding to the whole slide, however no binding to the antigen was detected. This doesn't come as a surprise since the antigen appeared to be denaturated. Anti-GFP showed specific binding to GFP.

iRIf picture of anti-GFP binding step - correct binding to the stamped GFP spot

iRIf picture of anti-salmonella binding step - unspecific binding to the whole slide

15.06.2015

2 Slides with APTES + PDITC (which were prepared from surchem 2 days before) were measured: Slide 306 and new slide (i9). Same experiment like before was conducted: each slide with 2 stamps, 1 iGEM stamp (100 ug/ml bBSA) and 1 Dia-Chip stamp (50 ug/ml GFP). Incubated 30 mins, pipetted PBS for 5-10 mins before stamping. Stamped and then preblocked 30 mins with 50 ug/ml BSA. Measured with 10 ug/ml StrepCy5 and 1 ug/ml Anti-GFP.

Slide 306: StrepCy5 step was only very weak in iRIf. Platereader afterwards aswell showed low binding. Furthermore, the shape of the stamp was kinda blurry.

When anti-GFP step was supposed to start the rack (CMMO) drove to a wrong position (approx. -0,5x, -0,5y). So no solution but air came into tube → canceled measurement. Slide i9: StrepCy5 was not seen in iRIf. Also antibody binding was not seen in iRIf. Platereader showed very weak Cy5 signal.

Slide 306: Cy5 signal in platereader after iRIf

Slide i9: Cy5 signal in platereader after iRIf

16.06.2015

2 Slides with GOPTS (which were prepared one day before) were measured: Slide 29 and slide 12. Same experiment like before was conducted: each slide with 2 stamps, 1 iGEM stamp (100 ug/ml bBSA) and 1 Dia-Chip stamp (50 ug/ml GFP). Incubated 30 mins, pipetted PBS for 5-10 mins before stamping. Stamped and then preblocked for a few hours with 1ml of 250 ug/ml BSA. Measurement in iRIf with 10 mg/ml BSA, 5 ug/ml StrepCy5, 5 ug/ml Anti-GFP

Slide 29: StrepCy5 step signal in iRIf was seeable, but weak. Anti-GFP binding could not be observed. Plate reader confirmed StrepCy5 binding. Slide 12: results were analogous to slide 29. Strep signal was weak, Anti-GFP binding not detectable. Even though blocking step showed properly the stamps. Platereader afterwards showed very strong StrepCy5 signal. We wondered why on the other hand it was quite weak in iRIf.

Slide 29: iRIf: Quotient picture before and after StrepCy5 step

Slide 29: iRIf: Quotient picture before and after Anti-GFP step

Slide 29: StrepCy5 signal after iRIf measurement

Slide 12: Quotientpicture iRIf before/after blocking

Slide 12: Platereader: Cy5 signal after iRIf

Additionally tests were conducted to investigate if proteins get pulled off, when blocking with the backside of a PDMS flowchamber. Half of each stamp was blocked with backside of a pdms flowchamber. Idea: When PDMS flowchamber pulls off the bBSA stamp, we should see a difference in signal intensity between the half which was blocked with PDMS and the one which wasnt. Stamps then were manually incubated for ~1h with 300 ul of 5 ug/ml StrepCy5. Intensity was observed in platereader: No difference between the halfes (blocked & unblocked) could be observed. Concluding from these experiments: Blocking with the backside of a PDMS flowchamber does not noteworthy pull of proteins (e.g. bBSA). →We should retry this approach to get rid off air bubbles when stamping.

Slide 216: Platereader signal after incubation of the stamps with StrepCy5

Slide 467: Platereader signal after incubation of the stamps with StrepCy5

Furthermore, our setup was tested. Measured Slide 208 (VisionPharma). General setup works fine (Jürgen fixed some stuff). Syringe first drove to wrong position, so many air bubbles came into system. Anyway measurement was continued. Blocking and binding of Strep could properly be seen.

Slide 208: Quotient picture before and after StrepStep

Slide 208: Platereader after iRIf

18.06.2015

Testing why a-GFP didnt bind to GFP:

2 plasmaactivated Slides (287, 303) were prepared and stamped. 50 ug/ml GFP (Dia-Chip) and 400 ug/ml bBSA (iGEM-Logo) on each Slide. After stamping 1 slide (303) was blocked with PDMS. Fluoresence of GFP was measured within fluoresence microscope. Surprisingly, on no slide GFP fluoresence could be detected. –> There seems to be no GFP; this would explain why we didnt observe a a-GFP binding to GFP in the last experiments. Obtained new GFP from Max (~1ml of 1mg/ml GFP → stock in the -20°C fridge ZBSA), retried same experiment with new slides (121, 215). 121 was blocked with the backside of a PDMS flowchamber. On both slides GFP could be detected, but quite inhomogeously distributed.

19.06.2015

3 identical slides (i1,i2,i3) of the following experiment were prepared: Stamped 3 different dilutions of GFP and bBSA on 1 Slide (so 6 on each slide) Concentrations were 50 ug/ml, 200 ug/ml and 500 ug/ml for either GFP (antibody stamp) and bBSA (stars-stamp / christmas tree-stamp) - Incubated 30-40 mins (50 ul) on the stamp, then stamped on the plasma activated slides. After stamping they were put in PBS. Fluoresence of all slides were measured. GFP signal was clear; slide 1 antibody for 500 ug/ml was missing (cause I screwed up stamping). Yet, intensity of GFP fluoresence was quite similar for all GFP concentrations (all measured with 3s shutter speed).

After measuring slide i1 was taken dried and blocked with backside of PDMS flowcell to see if GFP signal gets less (if blocking pulls of protein). Fortunately, GFP intensity was same before and after blocking with PDMS.

Slide i1: GFP fluorescence 200 ug/ml - before blocking with PDMS

Slide i1: GFP fluorescence 200 ug/ml - after blocking with PDMS

Slide i2: GFP fluorescence 50 ug/ml

Slide i2: GFP fluorescence 200 ug/ml

Slide i2: GFP fluorescence 500 ug/ml

Slide i3: GFP fluorescence 50 ug/ml

Slide i3: GFP fluorescence 200 ug/ml

Slide i3: GFP fluorescence 500 ug/ml

Slide i2 was measured (also blocked with PDMS flowcell). Binding of Strep to bBSA can properly be seen. Yet, intensity was expected other way around (becauses here: intensity of 50 ug/ml stamp > 200 ug/ml stamp > 500 ug/ml stamp). This may have 2 reasons: 1. Since the fluid (Strep) flows from left to right, all Strep molecules might accumulate at the most left stamp (50 ug/ml) and so constantly decrease from left to right. 2. 50 ug/ml stamp was the first stamp that was made on the activated slide; second stamp was done a little later (on a maybe less activated slide), which may lead to a decreased transfer of protein to the surface. This would maybe also be an explanation for the constant amount of GFP fluoresence (maybe higher concentration and less activated slide counterbalanced each other?!)

Slide i2: Quotient picture (before/after) StrepCy5; left to right: 50 ug/ml, 200 ug/ml, 500 ug/ml stamps

Slide i2: Quotient picture (before/after) Anti-GFP; left to right: 50 ug/ml, 200 ug/ml, 500 ug/ml stamps

Slide i2: platereader after iRIf (only 33% laser power!)

20.06.2015

Slide i3 was measured (also blocked with PDMS flowcell). Two things were changed: Slide was put other way around into flowchamber (so this time the 500 ug/ml stamp is flushed first… compare with pictures from slide i2). Secondly, the iRIf protocol was slightly changed due to the fact that anti-GFP (mouse) didnt bind yesterday. Protocol now: (blocking BSA), StrepCy5, anti-GFP (mouse), anti-GFP (goat, biotinylated), StrepCy5

Slide i3: Quotient picture (before/after) StrepCy5 - first flush

Slide i3: Quotient picture (before/after) Anti-GFP (mouse, not biotinylated)

Slide i3: Quotient picture (before/after) Anti-GFP (goat, biotinylated)

Slide i3: Quotient picture (before/after) StrepCy5 - second flush

Slide i3: Platereader after iRIf. Cy5 intensity

As can be seen, StrepCy5 binding (first flush) was good, but a little weaker than for slide i2. Anti-GFP (mouse) again didnt bind to GFP, but Anti-GFP (goat) did bind properly to GFP. Second flush with StrepCy5 confirmed that the biotinylated antibody did bind to GFP (although signal is quite weak). Platereader signal afterwards showed intense Cy5 signal for the bBSA stamp and less signal for the GFP/biotinylated anti-GFP stamp.

Two plasma activated stamps were stamped onto a plasma activated slide. Flooding shows a few bubbles, the shape of the stamps are indistinguishible

11.06.2015 Two DiaCHIP stamps were cut out and plasmaactivated for 30 sek, then incubated with bBSA for ~60 min with 50µg/ml bBSA. They were then blocked for 15 min with 50µg/ml BSA. Afterwards they were then stamped onto a plasmaactivated slide (S29) and used in the iRIF setup. The stamping took 30 sek each. One stamp was pushed very gently onto the glass slide, the other one was pressed onto the surface as always.

Observations: Incubation with bBSA showed that the PDMS is a lot more hydrophilic after plasma activation. Normally we incubate with 50µl solution, however with the plasma-activated PDMS, the solutions flows down the side of the stamp after about 30µl. Flooding in the iRIF showed bubbles, however they don't seem as bad as usual, since the shape of the stamps can't be recognized. The stamp above was gently pressed onto the slide, the lower one with regular pressure.

12/13.06.2015 stamped 4 slides with plasma activated stamps. Stamps were all hydrophyllic. However in no case did strep-cy5 bind to the stamp. Fluorescence never showed any signal, indicating that the protein is bound to the stamp and does not dissassociate. Slide 314 was damaged because PDMS bound to the slide and stuck to it. Even after rubbing heavily, the PDMS remains on the slide. Conclusion: Activated stamps are more hydrophyllic and show less bubbles when flooded, but are useless because the protein won't dissasociate from the stamp

Also retried non-plasma activated stamps. The stamps were pressed only were lightly onto the glass and then blocked for 30 min in 50µg/ml BSA solution. In both cases the stamps were flooded, however the bBSA/StrepCy5 signal was very weak - even though in one case a 5-fold strepCy5 concentration was used (50µg/ml) The experiment was thus canceled before the antibody step started to save some antibody. Fluorescence for the 50µg/ml StrepCy5 showes a strong fluorescence signal however.

Conclusion: Higher StrepCy5 concentration didn't improve iRIf picture

50µg/ml StrepCy5 solution was used. iRIf signal was weak, fluorescence quite strong

04.06.2015

2 Slides were prepared (314,24): Each with 2 stamps: 1 stamp 40ug/ml bBSA, 1 stamp 50ug/ml His-GFP. Slides were only plasmaactivated and then stamped. 314 was measured. 24 stored in PBS to be measured on next day.

Slide 314 was measured: after initiation problems with rack it was measured, unfortunately air bubbles came into systems (maintained on the bBSA stamp). However, binding of anti-GFP to GFP could be seen well (right stamp - vertikal to flow direction).

Meanwhile fluoresence of Slide 24 (replicate) was measured under fluoresence microscope. Suprisingly, it was seen well (concentration of GFP (only) 50 ug/ml).

05.06.2015

Slide 24 was meaasured in iRIf. Problems with device: Unstable flowcell evoked high noise; furthermore large airbubble came within the flow chamber. Again, anti-GFP to GFP binding could be seen anyways (right stamp).

Binding of Strep to bBSA could not be seen, probably because it didnt bind. Cy5 fluoresence was checked afterwards and showed that strep-cy5 barely bound to the bBSA stamp.

Slide 24 (05.06.15)

2 Slides were prepared (new iRIf slides): Each with 2 stamps: 1 stamp 40ug/ml bBSA (Gitterstempel), 1 stamp 50ug/ml His-GFP (iGEM-Stempel). Slides were only plasmaactivated and then stamped. Blocked for ~1h: pipetted 1 ml BSA(250ug/ml) on each slide and put on rotator. They were stored in PBS to be measured on next day.

06.06.2015

Slides which were prepared the day before were measured. First GFP fluoresence was checked out in microscope: were seen good in both cases (iGEM stamp). Then slide i1 was measured. Binding of antiGFP to GFP was incredibly intense.

Surprisingly, also StrepCy5 bound to GFP/antiGFP: iRIf signal was seen on GFP stamp (iGEM-Logo) at StrepCy5 step. Plate reader after measurement showed an extraordinary signal in the Strep channel on the GFP stamp (iGEM-Logo), decent signal on the bBSA stamp (Gitterstempel)

Slide i1: Under fluorescence microscope before iRIf measurement

Slide i2: Under fluorescence microscope before iRIf measurement

Slide i1: during iRIf measurement within the anti-GFP binding step

Slide i1: during iRIf measruement within the StrepCy5 binding step

Slide i1: in plate reader to see StrepCy5 after iRIf

To investigate that phenomenon we conducted some experiments with the unmeasured replicate (i2). First we pipetted StrepCy5 manually on the bBSA and the GFP spot and incubated for a while: StrepCy5 only bound to the bBSA stamp. Then we pipetted anti-GFP to both spots and checked for Cy5 fluoresence. still only bBSA spot signal was seen. Then we pipetted StrepCy5 again on the stamp, to see if it binds to the anti-GFP, but again StrepCy5 signal was only visible on the bBSA stamp (Gitterstempel) and not on the iGEM stamp. We left kinda clueless why we saw StrepCy5 in iRIf and plate reader on the iGEM stamp with slide i1. *Edit: Since the anti-GFP antibody seemed to be biotinylated its not that surprising that strepCy5 also bound to antiGFP

Slide i2: plate reader after pipetted manually strepcy5

Slide i2: plate reader after pipetted manually strepcy5, anti-Gfp and again strepcy5

06.06.2015 2 Slides were prepared (new iRIf slide [i4], slide 287): Each with 2 stamps: 1 stamp 40ug/ml bBSA (antibody stamp), 1 stamp 50ug/ml His-GFP (Dia Chip stamp without antibody). Slides were only plasmaactivated and then stamped. Blocked for a few hours: pipetted 1 ml BSA(125ug/ml) on each slide and put on rotator. They were stored in PBS at 4° to be measured on next day.

07.06.2015

Slide 287 was measured. When chamber was flooded airbubbles stuck at the end of the stamps. Slide was additionally blocked with the backside of a pdms flowcell. After this no air bubbles occured (but probably the pdms flowcell pulled of some (or a lot) of the stamped protein). No iRIf signal for the StrepCy5 to bBSA stamp binding could be observed. Signal for anti-GFP/GFP binding (DIA-Chip stamp) was good. Plate reader after the measurement confirmed that StrepCy5 bound only slightly to the (probably largely pulled off) bBSA stamp. Plate reader showed very little unspecific binding of StrepCy5 to the GFP/antiGFP stamp.

Slide 287: Plate reader after iRIf measurement

02.06.2015: 4 stamped slides were prepared the day before (plasmaactivated, stamped 40 ug/ml bBSA, preblocked ~2h with 500 ug/ml BSA, stored o/n at rt)

2 slides (Gitterstempel) were measured. Slide 314 was measured without problems. But no iRIf signal was visible. Even though the binding did happen as seen in the plate reader. Slide 29 (Exposure time was increased to 15ms) airbubble came into system. Continued measuring but saw no signal in iRIf, but stamp was also only hard to see in plate reader.

Slide 121 (Logo) was measured. Strep-Cy5 with 50ug/ml concentration. Large air bubbles came into system after blocking step, but continued measuring. iRIf signal was visible. Yet, still bad quality due to high noise of the picture and inhomogenous illumination. Plate reader showed very intense and well shaped logo → For experiments to just show our logo we should use high concentrations of Strep-Cy5/antibodies.

02.06.2015: SurChem prepared two slides (one GOPTS, one APTES/PDITC) and spotted 0,5 µl spots (without PDMS spotting aid): GFP-stock (1,67 mg/ml), GFP (1:500), GFP (1:1000), 2x bBSA 2µM

The new slides were used and thus have no number yet. Surface chemistry of the GOTPS slide might be on the wrong side. iRIf measurement with 5 µg/ml anti-GFP and 5 µg/ml Strep-Cy5. Measurement was performed on both machines in parallel. Both problems with both new slides, water escaped from the flwo cell - it might be possible that the pdms flow chambers don't stick as well to the new slides or the new surface chemistry

Light in the GOPTS measurement was overexposed.StrepCy5 was not flown into the chamber due to an error in typing the correct eppendorf position in the excel sheet. Anti-GFP spots might be visibile, but cant be safely confirmed

APTES/PDITC might either spotted or measured the wrong side. Strepcy5 was not observed in fluorescence, therefor the huge iRIf files were not analyzed jet.

31.05.2015: Slides 24 & 314; Plasma-activated; Spotted with 15µl (50µl/ml gfp; 40µl/ml bBSA) and PDMS spotting-aid. Incubated for 30 min, then blocked with BSA+PDMS for 30 min; After blocking, the slides were put in contact with the back-side of a PDMS flow cell to avoid problems with hydrophobicity

iRIF measurement on right-side device (ours was broken again): anti-gfp 1:1000 first, then StrepCy5 (5µg/ml)

Results: fluorescence of S24 and 314 shows both Cy5 spots are visible (+one unspecific binding spot in the case of S24). Anti-GFP binding could not be observed in iRIf. GFP could hardly be seen in fluorescence microscope ⇒ GFP concentration was far to low, SurChem uses 1 mg to spot!

Fluorescence microscope image of a GFP spot. Shutter time 8s

30.05.2015: Slides: 121, 287 & 314; Used Roths setup; Plasma-activated, then stamped with 50µg/ml (50µl) bBSA; after stamping, blocked with back-side of PDMS flow-chamber, then blocked for 30min in BSA+PBS

S287 with Flowchamber I1: first try failed because rack did not move, flow chamber showed two bubbles. Second try showed no bubbles, flooding of chamber not present in measurement (camera not running) S121 worked without problems. S314 was not blocked, but only incubated with PDMS after stamping. Showed bubbles in flooding step

Slide 121, stamped with bBSA, then blocked with PDMS flowcell, then blocked with BSA - Roths setup was used

Slide 121 - corresponding iRIf Quotient picture of slide 121

S121 - fluorescence and iRIf comparison

Slide 287, stamped with bBSA, then blocked with PDMS flowcell, then blocked with BSA - Roths setup was used

Slide 314 - blocked without BSA, only in contact with PDMS - little unspecific binding, but less signal than S121 and 287

23.05.2015 Slides used: 303, 314, Plasma activated. Stamp: „the grill“-stamp. bBSA was stamped, binding with Strep-Cy5

S303 was stamped with 40 µg/ml bBSA, S314 with 400 µg/ml; After stamping, the slides were put in a petridish and covered with BSA and PBS for 30min under rotaion. Flowchambers used: I1 and I2 (both closed fine)

S314: the last PBS step pumped air into the chamber because the PBS-falcon ran dry during the measurement. The Strep-Cy5 incubation prior to it worked fine though. (The machine still did not work as it should have → too little BSA/StrepCy5 were sucked in by the syringe, rack had some problems before measurement). Fluorescence shows that the binding worked, the stamp was therefor flooded correctly. iRIf results are hard to obtain due to the air entering the chamber in the last step.

S303: was accidentally touched while it was put into the chamber → no binding occured, fluorescence shows that the stamp was completely smeared.

Conclusion: the problem might be our stamp, since the „grill“ stamps are flooded correctly.

Additional hydrophobicity tests

After measurement PBS was put on the slide 314 (the whole slide was blocked with BSA prior to measureing). The buffer took the shape of the flow chamber, showing that PDMS is hydrophobic and that the PDMS remains on the slide. This behaviour persisted, even after washing with SDS, EtOH, Aceton, Water. After adding NP40 or dish washing soap to lower surface tension, the water remained in the shape of the PDMS flow chamber on the slide, even when inclinating the slide. The PDMS shape disappeared after plasma activation of the slide

Slide 314 after measuring in iRIF. PBS was put on slide and takes the shape of the flow chamber

24.05.2015 Stamping „the grill“ on plasmaactivated slides without preblocking them after stamping. Results: The flooding appears to be worse in unblocked slides than in BSA blocked ones. We see bubbles around the stamps, mostly in the „Windschatten“(n=3)

Testing whether blocking changes the hydrophobicity of the PDMS diamonds We observed that in slides without preblocking, the flow-cell diamonds were surrounded by bubbles. To see whether this still occurs in blocked slides, we used a diamond-flowcell on a blocked, plasmaactivated slide.

⇒ Conclusion: The bubbles showed up on the blocked slide. When the flow cell is changed into a flowcell without diamonds, the bubbles will still adhere to the spots where the diamonds touched the slide. Blocking therefore does not reduce PDMS induced hydrophobicity.

Blocked slide with a flowcell that has diamonds. Air-bubbles adhere to the diamonds

Zoomed version of air-bubbles adhering to the PDMS diamonds

The same slide, now on a flow cell that has no diamonds. The Bubbles remain, showing that the hydrophobic PDMS remain on the slide and hinder flow into the chamber

Testing flow-mechanics when the whole slide was in contact with PDMS Slides 121 and 314 were plasma activated and stamped. The backside of a flow chamber was put on S121 after stamping, to see whether we're able see a difference in how the cell is flooded if the whole slide is hydrophobic. For Slide 314 the back side of a flow-chamber was put on the slide before stamping.

Slide 121: The flooding worked fine, no air-bubbles stuck to the diamonds. The machine broke down after flooding, therefore we manually incubated with Strep-Cy5 and measured fluorescence. ⇒ Conclusions: The flooding of stamps works better if PDMS had contact with the whole slide. The stamped protein remains on the slide, even though a PDMS slide was put on top of it. This might be usefull when stamping difficult stamps (DIAChip logo)

Slide 314: The fluorescence was far lower and the slide hardly visible, which indicates that stamping after contact with PDMS is not very efficient.

24.05.2015 - S121 was stamped with bBSA, then the back-side of a PDMS flow cell was put on the slide. The flooding showed no bubbles sticking to stamp or diamonds. Fluorescence shows that bBSA did not disassociate when the backside of the flow cell was put on the slide

Slide 314 - The backside of a PDMS flow cell was put on the plasma activated slide, then it was stamped with bBSA (reverse order than S121) - little fluorescence visible, bBSA did not bind to the glass

20.05.2015 Slides 215(& 29) - Ni-NTA coated slides see surchem labjournal

Antibody: Anti-GFP Goat - MA5 15256 - Stock 1 mg/ml was diluted 1:10000 in PBS (100 ng/ml) His-GFP spotted Ni-NTA slides prepared by the Surface Chemistry group 3-4 days prior to our measurement. Before iRIF measurement, fluorescence was measured on the array scanner to see the Cy5 control spot. Spots were hardly visible in fluorescence (values had to be cranked up to absolute max!)

iRIf measurement failed for various reasons: the iRIf syringe did not suck up the solutions it was programmed to. The also rack did not move the way it was supposed to. We got air-bubbles because the syringe did not go far enough into the eppis and sucked up air which entered the flowchamber. Interestingly, the bubbles adhered exactly where the location of the spotts were.

Antigfp-gfp binding could not be observed due to the fact that the spots were covered by air-bubbles. Details can be seen on a movie made from the results.

⇒ Conclusions: We can't make any because the machine was broken.

Slide 314: Flourescence measurement - Strep-Cy5 bound fine, stamp was flooded without any major problems

2015.05.19: Measured 2 slides with 1 stamp logo stamp each (Dia Chip logo) Slides used: 303 & 314. Slides were stamped and then preblocked with 50µg/ml BSA solution for 30min. Flow chamber was also preblocked for approx. 30 min. New iRIF setup showed various troubles. Camera exposure time was wrong, as well as some other settings. Kinda got over that part eventually. Results: First slide (303) showed bubbles again. The measurement was stopped, then restarted with an extra 1%NP40 wash-step. Bubbles still persisted so we continued to measure anyways. Once the measurement time should have been over, the control software didn't show up that it had completed. Finally we realized that some solutions weren't used at all, or at least less was taken out than should have.. so maybe the „solution-wagon“ got stuck during the measurement.. we're kinda clueless about that step. UPDATE: The syringe didn't go deep enough into the eppis -_-

Slide 303 since the other slide showd bubbles, we tried to rub over the slide that was still incubating with a kimtech tissue and put new blocking buffer on top of it. Result: No bubbles showed up.. because there was no more stamp on the slide Conclusion: don't rub with Kimtech tissue on stamps of plasmaactivated slides

20.05.2015 - iRIF measurement of His-GFP spotted on slide. Incubated with anti-gfp antibody. Air entered the flow cell due to a broken setup. Interestingly, the bubbles adhere to the spot-locations

2015.04.14: Plasmaactived slides 215 & 314 were stamped with grid-stamp. Each slide was stamped twice. One stamp with 500 ug/ml BSA, the other with 50 ng/ml (1:10000) to see if iRIF detection is still possible after strong dilusion. 1:10000 was barely visible in iRIF, but still better than under fluorescent array scanner. Forgot to take fluorescence pictures of slide 215. Results under 150414_314_P_bBSA_StrepCy5 and 150414_215_P_bBSA_StrepCy5

2015.04.01: Slides used: Slide 1: BW-A-01-002-315 Slide 2: BW-A-01-001-314 Results saved under 150401_APTES_bBSA_StrepCy5_S314 Measured two APTES-PDITC slides - spottet with bBSA - produced by the surface chemistry group the same day. Slides were left in the fridge for approx. 1h. We encountered several problems putting the glass slides on the flow chambers as they constantly started to collapse. Flowchambers and slides were changed, but the problem persisted. After approx 1h we were lucky and able to measure the slides.

Results: BW-A-01-002-315 ⇒ The silhouettes of the spotted bBSA were visible in the iRIF measurement, however no binding of Strep-Cy5 could be detected. Fluorescence measurement after the iRIF measurement showed however that Strep-Cy5 had indeed bound to the spotted areas. Spotting was therefore successfull, the iRIF measurement failed however for unknown reasons.

BW-A-01-001-314 ⇒ iRIF measurement was successful as the spots were visible both in iRIF and fluorescent imaging. In both slides the spots were smeared.

⇒ Conclusions: both APTES+PDITC slides were able to bind Strep-Cy5, our APTES+PDITC protocol seems to work. iRIF measurement failed on one slide, the reason remains unknown.

01.04.2015 - BW-A-01-001-314 - bBSA was spotted on APTES-PDICT slide and strep-cy5 bound to it in the flowchamber. left: fluorescence measurement; right: iRIF measurement

2015.03.27: Tested printing bBSA directly on three activated TaO₅ glass slides (without GOPTS). iRIF measurement failed because one of the tubes was congested. Two of the glass slides remain in the fridge for measuring after the weekend.

2015.03.30: Continued the measurement on the residual two rif-slides. Slide S303: stamped bBSA visible via iRIF, though some bubbles entered the flow-cell. Fluorescence measurement revealed that the stamps looked fine. Results are saved under „150330_bBSA_StrepCy5_S303“. Slide 024: tried two times, both times the cell (probably) collapsed because some input flow might have gotten behind the PDMS flowcell. iRIF results we're not saved since nothing could be seen.

⇒ Conclusions: iRIF worked for at least one slide. We'll have to try one of the two new flowcells prepared by our team to prevent further collapse. Also the stamp appears to have been partially dried out before stamping, a faster transition from protein-incubation and stamping is advisable.

30.03.2015 - iRIF of bBSA+StrepCy5 on activated slides

30.03.2015 - Fluorescence measurement of bBSA+StrepCy5 on activated slides