GST fusion prot isolation

Duncan Clark Duncan at genesys.demon.co.uk
Sat Jan 15 05:28:44 EST 1994


>> From: Elaine M. Weidenhammer <ew2f+ at andrew.cmu.edu>
>> To: methods-and-reagents at net.bio.net
>> Subject: GST fusion prot isolation
>> 
>> My hypothesis is that the
>> detergent in the buffer I'm using to extract the pellet is inhibiting
>> binding to the beads.  Does this sound reasonable, and, if so, does
>> anyone have a protocol that will enable me to isolate my protein without
>> taking large losses of yield?? Thanks
>> elaine

Elaine,

This is perfectly reasonable. In Dec 93 Biotechniques pp989-992 a solution to 
this problem is given. Basically you need to get rid of the detergent. Try 
solubilizing with 0.5% SDS and ppt the SDS from the supernatant with 140ul 
1M KCl/ml supernatant. Incubate at 4C for 30 mins then microfuge etc You must
remove the KCl by running down a Sephadex G-50 in say PBS. This should then
allow your coupling to occur at high efficiency. 

Duncan
-- 
-----------------------------------------------------------------------------
Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
-----------------------------------------------------------------------------



More information about the Methods mailing list