2 replies to this topic

[Ashish Saroha]

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

[bob1]

How are you assessing the transfection efficiency?  I would be surprised if you are getting that high an efficiency using an endosome based reagent and if you re using another plasmid (e.g. eGFP expression) then you are not assessing the transfection efficiency of your plasmid, only the GFP expressing plasmid!

Could the gene product be toxic?  Have you looked for transcription of the RNA?  Have you tried using an anti-his antibody (Flag on Brit-tap is non-canonical, so may not be recognised)?

[gyma]

protein with 125 aa. is about 15kd. it is relatively small so you have to be careful when doing sds-page and transfer. are you sure it is not running out of the gel? and also are you sure it is not lost during transfer?
if these are all ok,  anti-his antibody may be another choice.