Transfection of a 230 Kda protein in HEK293

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RachelD
RachelD's picture
Transfection of a 230 Kda protein in HEK293

Hello everybody,

I'm trying to overexpress a protein of 230 KDa in HEK293. I have been trying for 3 months but I don't see anything on my western blot...
I use Lipofectamine, or Xtrem Gene ( supposed to be better)
I lyse with SDS2%
I use a 4-12% NuPage gel, and i transfer during 3 hours approximately
I use antibodies tthat have been validated on western before...
Any idea to optimize the protocol?

thank you very much,

Rachel

Jason King
Jason King's picture
 I usually freeze-thaw Lyse

 I usually freeze-thaw Lyse and then sonicate the sample using a high power probe (not a simple sonic action water bath). If this doesn't help, it might be worth spot blotting samples of the cell extract and doing the antibody detection on these. If this works it would suggest that the protein is not entering the gel.

i'm presuming that you haven't modified the proteins sequence in some way. If you have then check that if the antibody you're using to detect the protein is a monoclonal - it doesn't detect the epitope that you've just changed. If this is a problem then use a polyclonal antiserum instead.

PM

RachelD
RachelD's picture
Thank you very much for your

Thank you very much for your help.
I already sonicate with a high power probe, but i'm going to try spot blotting my samples.

Rachel