-> We need to know how ORF1p and ORF2p are expressed in each condition before doing I-DIRT or SILAC
A) Relative yield by IP from each tagged cell line
Start from 50 mg of powder for each condition
Extract 1:4 (200 ul) with extraction buffer – sonicate and collect the cleared lysate
– save 20 ml of SUP (if protein concentration is enough)
– save pellet (50 ml)
IP affinity medium αFlag (5 ul)
- save FT
Elute from affinity medium
- save 10 ul of elution for SDS-PAGE
Run SDS-PAGE – (ask about which samples run here)
- save 10 ul more for Wes
B) Bradford/BCA – Relative Protein Concentration
Determine relative protein concentration by BCA (use the same dilutions that were used for the antibody validation + testing)
C) Relative amounts of expressed ORF1 & ORF2 and OCT4 & FOXF2
Determine the expression of each of these antibodies in the 3 samples – PC-H-t, PC-L-ut and DC-H-t.
Antibodies (use the antibody dilutions used in the antibody validation + testing in DC spare batch and PA-1):
αORF1p
αORF2p
αT7
αFlag
αOCT4
αFOXF2