"Total Protein Extraction with SDS buffer (DC-H-t (B3), PC-H-t (B1), PC-L-ut (B1))
"
Date: 05/23/18
Cell lines:
DC-H-t (B3)
PC-H-t (B1)
PC-L-ut (B1)
Extraction/wash buffer: 2% SDS buffer,
40 mM Tris pH 8 (with protease inhibitors)
Scale: 50mg per cell line
Weigh out 50mg of powder/cell line, add 200ul of SDS buffer to each tube (1:4 w/v), vortex to mix
5x 2sec @ 2 Amp with the new sonicator
Western blot:
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