His tag protein purification problem - Binding of undesired protein (May/31/2006 )

Hi all,

I am new here. I am having problem with my his tag protein purification. My protein is about 115 kDa without the tag and I am using the Talon resin. I am getting undesired nuclear protein binding to the beads. Is there a way to prevent that. Moreover, what does the binding buffer comprised of? I am using a Wash/Extraction buffer for the binding and washing and the elution buffer with 500 mM immidazole for elution. Anyone can help?

I generally use Ni-NTA resin instead of Talon so I dunno if this is useful...

I'm assuming that you run SDS PAGE of your elution off column, how could you know that contaminating proteins are nuclear proteins? Are we talking about a whole bunch of contamination or just one or two contaminating bands on the gel (I've had a protein that binds another and they co-elute)?

I normally lyse cells in high salt (mM NaCl) + mM imidazole. Then pass lysate through an anion exchange column (protein should be in flow through) then onto Ni column. Column wash with 20cv buffer containing high salt + 30mM imidazole. then elute with 250mM imidazole.

Our lab uses Ni-NTA agarose from invitrogen to purify proteins with His tag.

if you are having problems with contamination try a multi step elution- i.e.50mM imidazole, then 100mM, 200mM and so on. Collect small fractions off the resin and you should find that the level of contaimination in the fraction containing your protein of interest is less. This is just based on the assumption that the other proteins will have a different affinity for the resin and should come off at a different imidazole concentration (whether that be before or after your protein).

We wash Ni-column with 20mM imidazole and elute with 250mM imidazole. But try to elute atleast 2 times, as we found higher protein conc. with 2nd elute.

在bufer中加入少量咪锉可以减少非特异性结合.

hi,
i would suggest u to follow HOLIDAY method, he sounds logical and i think it is the right way to target ur problem. at the same time try to use 20mM immidizole in ur wash buffer.

gud luck
payeli.