his- & biotin- tag protein purfication
Posted 04 April 2011 - 07:29 AM
I am purifying a protein with both his-tag and Biotin tag from cell lysate in denaturing condition (8M Urea & 1%SDS). I did his- pull down first and then biotin pull down. His pull down works fine with the his-tag Dynabeads. However, I got no binding in the biotin pull down.
His-tag pull down elution buffer: 8M Urea, 1%SDS, 10 mM DTT, 1 M imidazole in PBS with protease inhibitor
Dialysis against with binding buffer of biotin pull down: 3M Urea, 1M NaCl, 0.25% SDS in PBS, pH8.0, (2h in 4 degree)
Then I did the biotin pull down with Neutravidin-agarose beads, which I had successfully done without his-tag pull down. But this time it didn't work at all.
Two things I am suspecting & considering to change:
1. use EDTA instead of imidazole to do the his-tag elution, as people suggest that imidazole is likely to cause precipitate problem in dialysis (althought I didn't see it in my case).
2. skip the dialysis step as it may cause loss of the protein. Instead, dilute the eluate of his-tag pull down directly with lager volumn (10X?) of binding buffer. But I am not sure whether the imidazole/EDTA will have influence on the biotin-neutravidin binding.
Does anyone have any suggestions or comments on this?
Thanks in advance.
Posted 04 April 2011 - 12:08 PM
Posted 12 April 2011 - 11:28 AM
Also - there's the chance you're binding plenty of protein to the strept/neut. agarose and just not getting it off. The dissociation constant for those is around 10-14 (or something really high like that), which means you need harsh conditions to elute. Monomeric avidin binds much less tightly, and you can do a competitive elution w/ biotin or desthiobiotin.
Have you assayed your unbound and eluted fractions for your protein (blotting and probing w/ anti-His Abs will also tell you what might be happening to your protein)?