I tried different methods for celllysis after the expression of the recombinant protein (human interleukin-8). The expression works well but I don't have the protein in my supernantent after celllysis (E.coli).
I combine tris or phosphate buffer with 50mM NaCl and 0,2mg/ml Lysozyme (+1mM EDTA). I add proteaseinhibitors and DNase. After the 1.30h incubation on ice the samples are put into a sonication-waterbath for 30 min. After sonicationbath I make a frozen/thaw cycle with liquid nitrogen (30 min).
the supernatant doesn't have the recombinant protein. It's always in the pellet of the cellysat.
What is wrong?
A colleague told me that it needed higher salt concentration but the authors in many papers do also use between 40-200 mM NaCl. When i use more salt i can't use it for the cationexchanger. During or after Desalting it is going to precipitate. My protein should be soluble!
I guess i have inclusion bodies but I don't want to purify them - it's to difficult!!!
Please help me! I've been working on that sh... since april and i am totally frustrated.
I'll be happy for every answer.