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HIS elution: can imidazole be added after SDS-sample buffer?


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#1 klenow

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Posted 06 August 2012 - 07:06 AM

Hi,
I was doing some pull-down and IP and it seems I treated all samples equally and instead of doing an elution step I directly added SDS-sample buffer containing B-mercaptoethanol to "realease" the complexes from the beads.
The problem is that some of the pull downs were HIS-tag proteins (I used HIS-tag dynabeads from invitrogen) but I did not add any imidazol. When I have realised of this, I had already added the SDS sample buffer and heated the samples (95C, 5min). It is possible to add the imidazole after this or SDS+B-mercapto+boiling it enough to release the complexes?
Thanks a lot,
Best.

#2 mdfenko

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Posted 06 August 2012 - 01:39 PM

sds-bme-boiling should release the complexes and denature them. adding imidazole will make no difference at this stage.
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