MT302 - 50 mg scale IP with 3xFlag Dynabeads
Extract 1:4 (200 ul) with extraction buffer – sonicate and collect the cleared lysate
– save 60 ml of SUP
– 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
- save 10 ul more
Bradford with SUP (to check protein concentration)
BCA to quantify protein concentration for Wes (accurate)
Wes to test antibodies:
ORF1p, ORF2p, T7, FOFX2, 3xFlag
I did the Wes with these conditions:
However the Wes didn't work (we didn't see any clear bands corresponding with any of the expected signals). I don't show the Wes results because we can't see anything.
After this I tried another Wes, but using only HEK 293 lysates that Leila gave me (with concentrations of 0.4 mg/ml and 0.8 mg/ml). We still couldn't see any clear signal around the desired MW. It is necessary to test the antibodies to find the correct conditions for Wes.