ORF1, ORF2 and Flag IPs from PA-1 and MT302
Date: 06/20/18
Cell lines:
PA-1 cell pellet (50-100mg)
MT302
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 100 mg per IP reaction with 5ul of beads
Weigh out 100 mg of powder/cell line, add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix
2x 5x 2sec @ 2 Amp with the new sonicator
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the cleared lysate, save 25ul
Save the pellets; resuspend each pellet in 400ul of 2% SDS/40mM Tris, pH8;
IP @ 4°C for 30’
Save the flow-through
Wash 3x 1ml extraction buffer
Transfer beads to fresh tubes after 1st wash
Add 30 ul of 2% SDS, 40 mM Tris pH 8 and elute @ 70°C for 5’with shaking
Collect the eluate