Using monoQ ionic exchange column to seperate your protein from GST.
You will get very clean protein using NaCl gradient elution.
In article <36958B5A.C4032725 at leonis.nus.edu.sg>,
medp7060 at leonis.nus.edu.sg says...
>>Bernard,
>>Thank you for your reply.
>>I did check the digestion of my GST fusion protein by thrombin, at
least 80
>% was cut measured by the SDS-PAGE. Now I can get my protein off the
beads
>together with the GST itself and the GST-fusion protein by using higher
>concentration of reduced glutathione. The problem now is how to
separate
>them.
>> pI MW
>GST 6.19 27K
>my protein 6.24 46K
>>Any suggestions will be highly appreciated.
>>Dongxu, Singapore
>>Bernard P. Murray, PhD wrote:
>>> In article <3692E849.9D7687F6 at leonis.nus.edu.sg>, Dongxu Liu
>> <medp7060 at leonis.nus.edu.sg> wrote:
>>>> > Does anyone have the following problem as I ?
>> > I expressed my protein as GST-fusion protein which bound to
gluthione
>> > sepharose 4B. Unfortunately, my proteins still remains in the
Sepharose
>> > after thrombin digestion on the matrix.
>> > Are there any ways to recover my protein ?
>> > Thanks for help.
>>>> Assuming you can elute the undigested protein by some means
>> (GSH or other conditions) can you cleave the protein in
>> solution? If so you could do this and then purify your
>> protein away from the tag using the GSH-sepharose.
>> If the problem is poor digestion then it is worth
>> exploring a range of conditions. The suppliers of the
>> thrombin give fairly conservative initial conditions to
>> stop you shredding your protein but if there is little or
>> no cutting then try warmer, longer or a different buffer
>> (eg. add a little Ca2+).
>> I hope that this helps,
>> Bernard
>> --
>> Bernard P. Murray, PhD
>> Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
>
