Protein inhibitor(s) during immunoprecipitation ?

Dr. Stephen Hoare s.hoare at alexisuk.demon.co.uk
Mon Mar 18 12:06:54 EST 1996


In article <ijiwaru-1503962329140001 at dcn69.dcn.davis.ca.us>,
ijiwaru at wheel.dcn.davis.ca.us writes
>In article <4i9jpq$l8 at mercury.hgmp.mrc.ac.uk>, pdezoysa at hgmp.mrc.ac.uk wrote:
>
>> Dear Netters,
>> 
>> I would like to know if:
>> A) any of you out there have encountered degradation of target protein
>during immunoprecipitation (preliminary data indicates that the source of 
>> the proteolytic activity is likely to be the rabbit serum itself) and; 
>> B)also know which protease inhibitor (or coktail of inhibitors) could
>inhibit this protease activity?
>> 
>> Thanks in advance,
>> 
>> Priyal de Zoysa.
>
>Many, many moons ago when I used to do this sort of stuff, I remember
>using a cocktail of N-ethylmaleimide, phenylmethylsulfonylfluoride,
>aprotinin  with an option to add leupeptin.  This did a pretty good job of
>keeping proteases in check, although if you are going to look for enzyme
>activity, you may want to keep an eye on the N-EM.
>
>Lyle Najita
>Plant Pathology
>University of California - Davis

PMSF (phenylmethylsulfonylfluoride) is not a user-friendly reagent
(highly toxic). AEBSF (4-(2-Aminoethyl)-benzenesulfonylfluoride,
hydrochloride) also known as Pefabloc SC is a safer alternative which
can replace PMSF in most applications. It is used at the same
concentration as PMSF. Another big advantage of AEBSF is that it is
water-soluble and stable so that stock solutions can be prepared.

MW 203.23 . 36.46    C8H10FNO2S . HCl    CAS = 30827-99-7

It is available from Alexis Corporation (cat. no. 270-022).
 
If you would like more information, call or fax one of the numbers below
or e-mail me (s.hoare at alexisuk.demon.co.uk) or our US office
(alexis at primenet.com).
-- 
Dr. Stephen Hoare
Alexis Corporation (UK) Ltd.
TEL: +44 (0) 1949 836111
FAX: +44 (0) 1949 836222



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