LaCava Research Wiki

General LaCava Lab Protocol

Immunoprecipitation with antibody conjugated Dynabeads from cell powder
Before beginning, place sonicator arm in cold room so it can equlibrate to 4C. Precool benchtop eppendorf centrifuge. Fill cooler with LN2 and get a bucket of ice.

1. Precool labeled safe lock tubes in LN2. Also precool weighing tools and large tweezers.
2. Weigh out appropriate amount of cell powder (usually 50-100mg).
3. Return stock powders to -80C
4. Add RT extraction buffer with protease inhibitors added (1:4 w/v)
5. Allow samples to sit at RT for ~60 seconds before adding extraction buffer to avoid instant powder freeze. Mix by vortexing for 10 seconds. If powder is not fully resuspended return to ice to cool before repeating vortex.
6. Sonicate 5 x 2 sec at 2 Amp (NYC setting is 2 Amp, ERIBA setting is 4 Amp).
7. Spin @ top speed ~21k rcf, 4°C for 10’ (Eppendorf Centrifuge 5424R)
8. During the spin, wash beads (5ul per 50mg powder standard; 10ul per 50mg for antiORF1 beads) 3x in 1ml ice cold wash buffer
9. Transfer the supernatant to fresh tube; save pellets for optional analysis later; save 5% of supernatant for potential WB analysis
10. Set up IP reaction(s) with prewashed dynabeads; IP @ 4°C for 30’
11. Save 5% of flow through for WB analysis
12. Wash 3x 1ml cold extraction buffer; switch beads to fresh tubes at 2nd wash step
13. After third wash give beads a quick final spin to remove last ul of wash buffer
14. Elute the beads with 20ul 1.1x LDS, 70 °C for 5’ with mixing
15. Collect elution in fresh tubes
16. Add DTT to 50mM to all samples and heat @70 °C for 10’
17. Optional: save samples for gel in the -20c freezer
18. Heat the samples @70°C for 10’
19. Run 4-12% Bis-Tris gel to analyze the elution for gel and/or western blot analysis. If both, 90% for the gel and 10% for the western blot.