We expect to see ORF1 in every well that BTUB is observed
Cell line: N2102EP / 2A6 & HEK293 Flip-In Trex ~ at least 150 k
We validated our old secondary antibody using fluorescent Westernblot as explained here:
Validating fluorescent antibody using Westernblot and QuantLAS4000
Experiment design:
First round:
| Antibody | | Supplier # | | Organism | | Detection channel | | Final Target | | Storage | | Stock (mg/ml) | | Final (mg/ml) | | !Vol from Stock (ul) | | Final volume (ul) |
|---|
| Primary | | Novus #NB600-936 | | Rabbit | | - | | Beta-tubulinl | | -20 | | 1 | | 0.1 | | 8 | | 80 |
| Secondary | | Novus #NL004 | | Donkey | | 557 | | Rabbit IgG | | 4 | | 1 | | 0.1 | | 8 | | 80 |
Second round:
| Antibody | | Supplier # | | Organism | | Detection channel | | Final Target | | Storage | | Stock (mg/ml) | | Final (mg/ml) | | !Vol from Stock (ul) | | Final volume (ul) |
|---|
| Primary | | Abmart #5130 Millipore | | Mouse | | - | | ORFpl | | 4 | | 2 | | 0.1 | | 4 | | 80 |
| Secondary | | Novus #NL008 | | Donkey | | 637 | | Mouse IgG | | 4 | | 1 | | 0.1 | | 8 | | 80 |
Protocol:
- Hydrate the chip for 10 mins
- Add 1mL of cell with suspension buffer on chip and let the cell settle for almost 10 mins. Checking chip under microscope for 15-20% occupancy
- Wash the unsettle cells with suspension buffer
- Pipette 300u L lysis/run buffer on one end of electrophoresis cell
- Place the chip side up in Milo and run the Milo with the recommended setting. Pour the remaining lysis/run buffer.
| TYPICAL MILO SETTING | | VALUE# |
|---|
| Lysis time | | 10 seconds |
| Electrophoresis run time | | 1 minute |
| Electrophoresis voltage | | 240 V |
| UV exposure times | | 4 minutes |
- Remove the chip and wash in wash buffer for 2x10 mins
- Prepare first primary. Dilute 1:10.
- Remove the chip from wash buffer and tab it slightly, then place in spinner for 3 seconds
- Place the chip down on the antibody probing chamber. Incubate the chips with primary antibody for 2 hrs @RT.
- Wash 3x10 with wash buffer on shaker.
- Prepare first secondary antibody. Dilute secondary antibody 1:10.
- Remove the chip from the wash buffer and tab it slightly, then place in spinner for 3 seconds.
- Place the chip down on the antibody probing chamber. Incubate the chips with secondary antibody for 1 hr @RT.
- Wash 3x15 with wash buffer on shaker.
- Rinse the chip 3 times with DI water and remove the wash buffer and spin it for 3 minutes.
- Scan the chip
- Wash the chip 3x15 in wash buffer and repeat the steps from 5 to 16 with the second set of antibodies