Size-Exclusion

[WS]

Hi Jan.

Actually, NaCl (as all small ions) should distribute evenly across the
column when it has been equilibrated. Can you perform online
conductivity monitoring? - You should be able to "see" the constant
salt concentration in the solvent.
Another issue might be that your protein becomes too concentrated and
then precipitates and/or denatures. In that case, try to work at a
higher dilution or -if appropriate- try to optimize salt
concentration, pH or add eg urea (maybe also sucrose or maltose will
do, try to use something that does not disturb your activity assay) in
order to stabilize your enzyme.

Best reagrds,

Wolfgang

On Dec 10, 11:50 am, "Jan R. Krähling" <J-R.Kraehl... from tu-
braunschweig.de> wrote:
> Hello!
>
> We are trying to establish the sixe-exclusion chromatography in our lab to purify the enzyme of our interest. In the purification-steps before the  size exclusion we observe that a NaCl-concentration of 1M stabilize the enzyme better than lower NaCl concentrations. So we tried the size exclusion at first with a high salt concentration, but the enzyme wasn't detectable. At lower concentrations the enzyme is detectable and active. My question is, if it is possible, that through the size-exclusion the salt-concentration increased to such a high level, that this will damage the enzyme. Is it possible that the elution of NaCl is so strong delayed, that at the top of the column unexpected high concentration of NaCl occur?
>
> Thanks for your help!
>
> Jan