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remove BSA from IgG prep


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#1 jon

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Posted 10 November 2004 - 04:43 PM

Hi,

i am trying to purify a rat monoclonal antibody from tissue culture sup (1%FBS). after ammonium sulfate precipitation (50% fraction) i resuspended the pellet in PBS and dialysed against PBS to get rid of the salt. on SDS PAGE it looks like it has so much of albumin in it.
i tried affi-Gel blue gel from bio rad but it is not working well for some reason. i did equilibrate my sample in phosphate buffer. with proteinG, the yield was very low when i tried directly with TC sup. probably not all of them got eluted even after repeated elution. gel filtration (sephadex G100) does not separate them far enough. i have not tried ion exchage.
any suggestions?
thanks in advance.
-Jon

#2 Oleksii

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Posted 11 November 2004 - 07:53 AM

Hi,

Try protein G or A Sepharose from Amersham-Pharmacia. It should bind just your Ig, but not other proteins.

#3 biplabbose

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Posted 17 December 2004 - 02:01 AM

Its alwasy a problem in case of purification of antibodies from cell culture soup. Even if u r using a protein G or A column u can not totally get rid of the BSA. Its better to use a serum free media system. If you can not have a serum free media system, u can at best use SYNSER (a hybridoma growth suppliment) to replace serum.
best of luck
Biplab
http://www.biplabbose.wb.st




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