Iīm an absolute beginner with regard to protein purification, but want to do some traditional protein clean-up (Anion/Cation EX; Gelfiltration).
Iīm having tremendous problems with the first step: an anionexchange column (1ml Q XL from GE).
Whenever I load a cell lysate sample (~20mg, 2ml sample volume) nearly everything is in the flow through fraction and nothing really binds to the column as judged from WB and the chromatogram (I run: 1. 6ml 50mM NaCl buffer, then 15ml with a gradient form 50-1000mM NaCl; flow 0,5ml/min).
I donīt want to go below 50mM NaCl, cause Iīm actually interested in a complex my protein of interest forms. But regardless of the 50mM more proteins should bind to the column initially...
Before adding the lysate on the column, the lysis buffer (150mM NaCl, 0,25% Deoxycholate, 1%NP40, etc, no SDS ) is exchanged to start buffer with a desalting column ( also tried dialysis).
Iīve also tried Glycerol in the buffers with the same result.
Can anyone give me a hint how to get my proteins to bind???
Thank you very much in advance!
post also posted in "Biochemistry"
Edited by mirc, 27 May 2010 - 03:52 AM.