sticked protien - (Nov/21/2008 )

hi all,
I work with a his tagged protein . The protein sticks to Ni-NTA. It 's not coming out of the resin.we r using 6M guanidine hcl in lysis buffer and 8 M urea in wash and elution buffers in a ph range of 6.3,5.9 & 4.5.we used 50 mM edta,sds also but still protien is sticking thr . Can amy body help??

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have you tried imidazole to elute your protein?

if you use sds then you have to boil the beads.

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hi thr,
i use 200 Mm imidazole also but no use shall u tell in ur lab which lysis buffer and wash buffer ur using to elute protien

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