Hi,
I have an animal cell pellet in a 2 ml Eppendorf tube. Pellet is occupying 40 micro lit space in the tube (If I add 40 miL water in a tube, how much place the water is occupying the same amount of space this pellet is holding --- I am justing elaborating for removing any kind of confusion).
I am going to use Hepes lysis buffer, followed by sonication and later adding 3x dye.
Now, my question is how much lysis buffer I should use to have the best cell lysis?
About 5 to 10 volumes of buffer should be fine. Although, most companies reccommend larger volumes of their buffers. But it largely depends on the downstream processing of your sample. For SDS-PAGE you would want a slightly more concentrated lysate than for Immunopreciptation. The optimal concentration of protein in the lysate, according to Abcam, is about 1–5 ug/uL
I saw in Abcam protocol- they mentioned 300 miL for 5 mg pellet which is much more for SDS-Page. 5-10X looks good to me.