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Hi all,
I am interested in over expressing c terminal 125 aminoacids of my protein in HEK 293 cells. For this I m using pBRIT-TAP vector to clone my sequence. This vector has 6x His follwed by 3x flag c-terminal tags. Sequence is properly inserted and is in right frame with vector as observed by sequencing.When I m checking the protein expression in HEK 293 cells after 24 or 48 hrs,I m not able to see the over expression in whole cell lysate by Western blotting using anti-flag antibody.Even after enriching the lystae using flag M2 beads, I m unable to see the protein. For transfection I am using six well plates with 300,000 cells per ml, 3ug of DNA and 6ul of jet PEI reagent. The transfection efficiency is > 80%.
Waiting for your suggestions
regards
Ashish Saroha
