Streptactin sepharose purification- binding problemStreptactin
Posted 16 December 2011 - 04:27 AM
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
Posted 16 December 2011 - 07:34 AM
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).
genius does what it must
i do what i get paid to do
Posted 17 December 2011 - 01:47 AM
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.
Edited by Aj265, 17 December 2011 - 01:49 AM.