u7k0201 at sunmail.lrz-muenchen.de
Thu Sep 14 04:53:40 EST 1995
1) Your inhibitor is maybe attached with the "wrong" sinde, i.e. the binding site for your
enzyme to the agarose
2) or your enzyme runs through the coulumn despite tha fact that the column is appropriate
(because there's too much salt in the solution or the pH is "wrong").
1) desalt your your conditioned medium, eg. by dialysis or by using a desalting column,
then try if you are able to precipitate your enzyme with the modified agarose. If not, try
to change pH, buffer, ... (The Pharmacia company has a nice small booklet they give upon
request on this topic)
2) if nothing of 1) will work, try another acivated agarose that will bind other functional
groups of your inhibitor. Maybe NHSactivated sepharose (small prepacked cheap 1 ml-columns!)
from Pharmacia or similar products from BioRad will do)
2a)if everything failes, try other media like heparin-sepharose. I made quite good experience
with this (heparin is used as an affinity reagent for growth factors, but IMHO, it might
act as a cation exchanger). Try ANYTHING you can imagine: Phosphotyrosine or phosphoserine
might be interesting as well.
3) If you finally succeed in getting stuck your protein to your columns, try to elute from low
salt (e.g. 10mM tris buffer) to high salt buffer (0.5 or 1M NaCl e.g.)
Hope this helps!
(not connected with any company mentioned)
sg at nwu.edu (Stephen Gately) wrote:
>We are trying to purify an enzyme from serum-free conditioned medium. We
>have tested a variety of exogenous inhibitors to block the enzyme
>activity, and have found a potent inhibitor that is also available bound
>to cyanogen bromide activiated agarose.
>Despite blocking 100% of activity when the inhibitor is added exogenously,
>passing the conditioned medium through a column containing the inhibitor
>agarose, or batch adsorption does not block the enzyme activity.
>We would appreciate any useful guidelines for affinity purification, ie.
>salt, pH etc., or specific comments on why we observe the difference
>between exogenous inhibitor and bound inhibitor ability to block
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