Team:Freiburg/Labjournals/irif/Before July
iRIf Labjournal before July
Testing different surfaces and stamping approaches
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.
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.
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.
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.
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 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?!)
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
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.
Testing stamping with plasma activated PDMS stamps
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
Stamping bBSA and GFP, show anti-GFP-binding
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.
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)
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
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.
Trying out our device (left)
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.
Spotting GFP on APTES/PDITC and GOPTS
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.
Spotting GFP (with PDMS blocked slides)
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!
Stamping our DIAChip logo with PDMS blocked slides
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
Finding out the reason why our logo stamp isn't being flooded
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
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.
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.
Testing Anti-GFP binding to His-GFP spotted slides
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.
Practicing Stamping of our logo in new machine
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
Practicing Stamping on plasma activated slides
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
Testing APTES+PDITC
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.
Preliminary Testing Phase
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.
