Streptactin sepharose purification- binding problem - (Dec/16/2011 )

Hi,
I am trying to purify an archaeal protein expressed in E. Coli so I am using high salt buffers:
Lysis & wash buffer 2M KCl, 100mM Tris
Elution bufffer 2M KCl, 100mM Tris,destiobiothin 2.5mM
All buffers at pH 8.
I am using IBA Streptactin Sepharose and gravity flow colums.
I did one purification as described in the IBA manual and washed column with 6x1ml of wash buffer.
However, I got my protein and other that bind not specifically so I decided to do a 2nd purification and wash the column 2x10 ml of wash buffer.
In both cases I eluted with 6x 0.5ml.
Before the second purification I incubated the sepharose resin with lysate for one hour with rotation.
While in the manual they say not to do it I think in this case this could not have caused problems since the high salt would already inactivate proteases.
The problem is that when I washed the column more I did not recover anything. (no bands on SDS-PAGE)

I can't think what might be the cause of this.

Thanks for any ideas

did you look at the flow through and wash to see if the protein didn't bind in the first place?

did you regenerate the column between uses?

why do you think that high salt will inactivate proteases? it doesn't inactivate any of the proteases with which i used to work.

iba recommends working at physiological salt concentrations (ie pbs).

Hi,

Thanks for your reply!
Yes I ran flow through on SDS and the protein was there but I didn't have it in the washes in any detectable amount.

After the purification I had VERY weak bands in elution fraction 3 so that would also suggest that the protein was binding extremely weakly.
I am using such high salt because this is a protein from haloarchea so I want to extract it in a buffer with composition similar to its natural cellular environment.
I did use a regenerated column but it was regenerated only once. I basically regenerated the same column that
I used for the first purification of the same protein. also, I did the regeneration myself and based on the colour change of the regeneration buffer that worked perfectly fine.

As regards proteases I would expect such high salt to inactive them. Even if this wouldn't be the case they wouldn't degrade absolutely everything so I should still see something.