Problems with his-tag protein purification

Songkhla Chulakasian @Songkhla_Chulakasian

04 April 2010 0 3K Report

I did the expression of my target protein from pET24a (containing His-tag at C-terminal) with BL-21 bacteria and the expression level seems okay.

BUT... the his-tag recombinant protein could not bind with Ni-NTA column during protein purification step.

SDS-PAGE from the flow-though solution (after incubation with Ni-NTA) showed that my protein totally leaked out (I re-checked by western blotting)

I did adjust pH of buffer system before start the purification. Also, I tried the Osmotic shock (http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html) before cell lysis but it didn't help....

Please kindly suggest me what should I solve this problem....

Thanks you guys in advance

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