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His protein expression problem


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#1 habibul

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Posted 15 December 2009 - 11:17 PM

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.
thanks
habibul

#2 Vini

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Posted 16 December 2009 - 01:53 AM

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....




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