I am trying to lyse cells (HEK and CHO) for protein (recombinantly expressed). I've ran an SDS-PAGE using cell samples in 2% SDS and clear band showing my protein being expressed (quite a lot), but when I try using some lysis buffer, and centrifuged them - supernatant was ran on gel and the band was not there.
So far, I tried using (1) M-PER, (2) 150mM NaCl, 50mM Tris with 1% detergent (NP-40, Triton X-100, Tween 20 and Tween 80) (3) 50mM sodium phosphate, 300mM NaCl, 10mM Imidazole with 1% detergent (NP-40, Triton X-100, Tween 20 and Tween 80)
None of the above worked. Wonder if there is any other way to lyse them effectively.
My final goal is to extract them in native form as I would like to isolate protein by His-Tag Isolation. I've run the lysed supernatant from above through His Tag isolation and can actually elute some of my protein. So my assumption is that my lysis procedure is not optimal. Is what I think logical?