His protein expression problem - (Dec/15/2009 )
i am dealing with expression of a His tagged protein.I am using pCRT7 vector and like to transform in BL(DH3)PlySs competant cell.I dont find good expression of protein and even if i get a poor expression but purification is very poor as many nonspecific bands are in there in SDS-PAGE.I use to use TALON beads for purification.
please let me give some idea on His tagged protein expression and purification
please advice me.
Hi, I use Ni-NTA beads for purification of my His-tag proteins. For poor expression, you need to standardize temp. conditions n IPTG conc., among other things.....To decresase the non-specific bands, u can try by increasing NaCl concentration to ~150 mM in your lysis buffer....