Daniel:Notebook/ComboLock/2016-6-2
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Antibody Hybridization Test[edit]
Time to test out the antibody binding protocol like I did with C-probes
Protocol[edit]
- Mix PLA Buffer
- Hybridization
- Split methanol-frozen cells into 4 (1.25 mL methanol each)
- Pellet cells via centrifugation at 600g for 3 min
- Add PLA mix and probes to create hybridization solution
- Mix 1 uL cell lysate with 3 uL Probe mix
- Incubate 1.5 hours at RT
- Add 200 uL 0.05% Tween 20 in PBS
- Wash twice with 0.05% Tween 20 in PBS
- Pellet cells at 600xg for 3 min
- Resuspend samples in 50 uL 1X PBS
- qPCR
- Set up qPCR reaction according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- VIM-AbTest1 Results 20160602.png
Results w/o labeling
- 20160602-Antibody Test qPCR Curve.png
Results with labeling
Reagent | Stock Conc. | Final Conc. | Dilution | Volume for 1 mL solution (uL) |
BSA* | 50 mg/mL (5%) | 0.10% | 50 | 20 |
Tween 20 | 100% | 0.05% | 2000 | 0.5 |
Salmon Sperm DNA | 10 mg/mL | 0.1 mg/mL | 100 | 10 |
EDTA | 500 mM | 5 mM | 100 | 10 |
PBS | 10X | 1X | 10 | 100 |
nfH2O | -------- | -------- | -------- | 859.5 |
uL PA Mix | uL PB Mix | uL PLA buffer | |
Sample 1 | 0.5 | 0 | 2.5 |
Sample 2 | 0 | 0.5 | 2.5 |
Sample 3 | 0.5 | 0.5 | 2 |
Sample 4 | 0 | 0 | 3 |
Sample | Lane | Cell Sample | Cells | 2X Kapa SYBR qPCR MM | 100 uM Primer2 | 100 uM Primer4-RC | 100 uM Primer6 | 100 uM Primer12-RC | H2O | Total Volume (uL) |
Sample 1 (PA) | A1 | S1 | 1 | 25 | 0.5 | 0.5 | 0 | 0 | 23 | 50 |
Sample 2 (PB) | A2 | S2 | 1 | 25 | 0 | 0 | 0.5 | 0.5 | 23 | 50 |
Sample 3 (PA+PB) | A3 | S3 | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |
Sample 4 (No Probe) | A4 | S4 | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |
No Template | A5 | NA | 0 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 23 | 50 |
Mismatch Primers | A6 | S3 | 1 | 25 | 0.5 | 0 | 0.5 | 0 | 23 | 50 |
No Primers | A7 | S3 | 1 | 25 | 0 | 0 | 0 | 0 | 24 | 50 |
Primers + Antibodies | A8 | NA | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |