Team:Freiburg/Labjournals/irif/July
iRIf Labjournal July
Measuring spotted ABs from rabbit/mouse/goat with anti-mouse/anti-rabbit
30.07.2015
Spotted:
| # | spot | Concentration |
|---|---|---|
| 1-3 | AB from mouse | 1.0 mg/ml |
| 4-6 | AB from rabbit | 1 mg/ml |
| 10 | AB from Goat (negative control) | |
| 11 | bBSA | 1.0 mg/ml |
| 7-9 | GFP | 1 mg/ml |
Flush protocol:
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer | 1 | 80 | 940 | 1x |
| BSA | 2 | 60 | 500 | 10 mg/ml |
| Buffer | 5 | 60 | 300 | 1x |
| Anti-Mouse | 4 | 20 | 900 | 5 ug/ml |
| Buffer | 5 | 60 | 300 | 1x |
| Anti-Rabbit | 6 | 20 | 900 | 5 ug/ml |
| Buffer | 9 | 60 | 300 | 1x |
| Anti-GFP(goat;biotinylated/mouse) | 4 | 20 | 920 | 5 ug/ml |
| Buffer | 3 | 60 | 510 | 1x |
| Strep-Cy5 | 10 | 20 | 900 | 5 ug/ml |
| Buffer | 11 | 60 | 300 | 1x |
Results: The secondary antibodies bound properly to their corresponding antibodies derived from mouse/rabbit.
Measuring HIV,HCV,Tet,Sal with polyclonal and monoclonal ABs
30.07.2015
Spotted:
| # | spot | Elution no. | Concentration |
|---|---|---|---|
| 1-2 | HIV(17) | 2 | 1.0 mg/ml |
| 3-4 | HCV(10) | 2 | 0.74 mg/ml |
| 5-6 | Tetanus(11) | 2 | 3.75 mg/ml |
| 7-8 | His-GFP | 1.0 mg/ml | |
| 9 | bBSA | 0.1 mg/ml | |
| 10-11 | Salmonella(15) | 2 | 6.7 mg/ml |
Flush protocol:
| 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 |
| Strep-Cy5 | 10 | 30 | 600 | 5 ug/ml |
| Buffer | 11 | 60 | 300 | 1x |
| Anti-Tetanus (mk: HYB 278-15) | 12 | 20 | 900 | 20 ug/ml |
| Buffer | 13 | 60 | 300 | 1x |
| Anti-Salmonella (1301 Elu2) | 14 | 20 | 900 | 1.6 mg/ml 1:4 |
| Buffer | 15 | 60 | 300 | 1x |
Results: Experiment was conducted in duplicate. Both measurements showed no antibody/antigen binding. Positive controls (GFP/anti-GFP, bBSA/Strep) performed well.
Measuring cell free expressed His-tYFP on Ni-NTA surface
28.07.2015
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 |
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
Measuring HIV, HCV, Salmonella, Tetanus (spotted) on Ni-NTA surface
24.07.2015
Spotting pattern:
| # | spot | Elution no. | Concentration | Antibody | AB-conc.[µg/ml] |
|---|---|---|---|---|---|
| 1-2 | HIV(17) | 2 | 0.36 mg/ml | gp41 DDX1306 | 20 |
| 3-4 | HCV(10) | Mix (1-3) | 0.13 mg/ml | 20 | |
| 5-6 | Tetanus(11) | Mix (1-3) | 0.55 mg/ml | HYB 278-01 | 20 |
| 7-8 | GFP | 1.0 mg/ml | a-GFP (goat;biotinylated) | 5 | |
| 9 | bBSA | 0.2 mg/ml | Strep-Cy5 | (Strep-Cy5) 10 | |
| 10-11 | Salmonella(15) | 2 | 3.2 mg/ml | a-Salmonella-(pIG15_1301) | 100 |
| anti-His (mouse) | 20 |
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.
Measuring HIV, HCV, Salmonella, Tetanus, GFP
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)
| Antigen | Stamped [µg/ml] | Antibody [µg/ml] |
|---|---|---|
| HIV (pIG15_1703) | 400 | 20 |
| HCV (pIG15_1003) | 400 | 20 |
| Salmonella (pIG15_1503) | 500 | 20 |
| Tetanus (pIG15_1103) | 500 | 20 |
| GFP | 200 | 10 |
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.
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.
| Antigen | Stamped [µg/ml] | Antibody [µg/ml] |
|---|---|---|
| HIV (pIG15_1703) | 400 | 50 |
| HCV (pIG15_1003) | 400 | 50 |
| Salmonella (pIG15_1503) | 500 | 100 |
| Tetanus (pIG15_1103) | 500 | 50 |
| GFP | 200 | 5 |
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…
Stamping Cell-free YFP (Promega Kit)
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.
Stamping unfrozen pIG15_1501 Salmonella antigen
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.
Stamping pIG15_1501 Salmonella antigen
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)
Testing stamping technique
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 pIG15_1501 Salmonella antigen
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.
