his- & biotin- tag protein purfication - (Apr/04/2011 )

Hi all,

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.

Mingwei

I'd go for number 2. Neutravidin-biotin is a very robust interaction.

Have you done any work to confirm if the protein is actually biotinylated? If you blot a small amount of your Ni resin eluate and probe w/ Neutravidin-HRP, you should be able to see if the protein is actually biotinylated. If it isn't, then you know where your problem lies. If it is biotinylated, then you need to check your streptavidin/neutravidin agarose.

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)?