ORF2 IP (DC-H-t (B3), PC-H-t (B1)) with anti-FLAG beads
Date: 05/23/18
Cell lines:
DC-H-t (B3)
PC-H-t (B1)
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul of anti-FLAG 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, save 3x FLAG elution for another experiment
BCA of supernatants.
Load 25 ug of protein for sup (load the same volume of pellet and FT) and a 10% of the elution (1.5 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
Stripping of the botton membrane
Block membrane during 2 hours – TBS-T 1X 5 % milk
Botton of membrane: 1:1000 PCNA 4ºC o/n incubation
3x10’ washes TBS-T 1X
1:10000 anti-mouse HRP 1 hour @ RT
