ORF1 IP (DC-H-t (B3), PC-H-t (B1), PC-L-ut(B1), PA-1, MT302) with anti-ORF1 beads
Date: 06/06/18
Cell lines:
DC-H-t (B3)
PC-H-t (B1)
PC-L-ut (B1)
PA-1 cell pellet (50-100mg)
MT302
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul of anti-ORF1 beads
Weigh out 50mg of powder/cell line, add 200ul of extraction buffer to each tube (1:4 w/v), vortex to mix
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 200ul 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 15ul of 2% SDS, 40 mM Tris pH 8 and elute @ 70°C for 5’with shaking
Collect the eluate.
BCA of supernatants.
Load 50 ug of protein for sup (load the same volume of pellet and FT) and a 20% of the elution (3 ul) on a 15-well 4-12% Bis-Tris gel. Run 200 V for 40 minutes.
Transfer protein to a nitrocellulose membrane. 70 V during 2 hours.
Block membrane during 2 hours – TBS-T 1X 5 % milk
Top of membrane: 1:1000 Flag 4º C o/n
Botton of membrane: 1:5000 ORF1 4 º C
o/n
3x10’ washes with TBS-T 1X
Top and botton membrane: 1:10000 anti-mouse HRP antibody 1 hour @ RT
