how to extract proteins from HEK293
Posted 24 February 2011 - 02:24 AM
I seeded 2X106 in a 100 mm dishes and transfected with 10 ug DNA.
Could you please suggest me a protocol. It is not clear to me if it is better to detach cells with trypsin before the lysis, or it is better to lyse them directly with the loading buffer. Somebody then sonicate while other load directly the gel. I have seen that people for different cell lines uses different methods but I need something that works well for 293 and that allow me to understand if the protein I'm looking for is effectivelly expressed.
Thanks for help
Posted 25 February 2011 - 10:39 AM
Posted 28 February 2011 - 08:46 AM
Thank you kfunk106 for your replay.
I agree with you that probably everybody has its own method and I will habe mine in the future but starting with somebodyes' else experience is for me a better starting point.
I have a couple of question. When you say ".....resuspend them in 3 volumes of lysis buffer" did you mean three times the volume of cold pbs used for scraping?
And when you say "......I then remove the nuclei by centrifugation at 1000xg for 5 min at 4 deg C, and remove the lysates (this is your cytoplasmic protein)" as lysate did you mean the supernatant?
Posted 01 March 2011 - 10:45 AM
No...I mean to resuspend them in 3 volumes of lysis buffer, which is composed of 25 mM Tris pH 7.4, 0.5% NP-40, 150 mM NaCl, and protease/phosphatase inhibitors
Yes, lysate=supernatant=cytoplasmic protein. pellet=nuclei.