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From: Paul Russo (Paul.Russo@lsp.lsbc.com)
Date: Wed May 16 2001 - 11:57:25 EDT

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Subject: RE: Misc - Question
Date: Wed, 16 May 2001 08:21:19 -0400
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Dennis

I would say your best bet is to run a 2D SDS-PAGE on your expressed proteins
since their sequence is very similar. Running a 1D gel would be difficult
since, like you said, their masses are so close. Having that extra
C-terminal region on one of the proteins should make it separate somewhat
easily on a 2D gel. After that you could do a western to locate the
proteins. A second gel could then be run, followed by blotting and some
C-terminal sequencing on both spots to discern one from another. Another
alternative would be to affinity purify both proteins with your antibody,
and then try some classic chromatography (HPLC, IEX, ect) followed again by
C-terminal sequencing (assuming of course you were able to separate the 2
proteins). Not sure which way would be easier. Hope this helps.

Paul

-----Original Message-----
From: Dennis Leveson-Gower [mailto:dleveson@bccancer.bc.ca]
Sent: Tuesday, May 15, 2001 12:25 PM
To: Recipients of ABRF List
Subject: Misc - Question

Hi everyone,

I have a problem and I thought I might be able to find some help in this
discussion group.

I am co-expressing two proteins in CHO cells and then trying to detect if
one or both of them are expressed. The problem is that the two proteins are
identical except for short peptide sequence at the C-terminus. I have
antibodies that react with both fragments but not with each individually.
The whole proteins should be approximately 50 and 53 kDa in size so
separation on Western would be difficult.

I need a way to take whole cells, break them open, and then see if both
proteins are being translated.

Thanks,

Dennis.

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<HTML><HEAD>
<META HTTP-EQUIV="Content-Type" CONTENT="text/html; charset=iso-8859-1">
<TITLE>Misc - Question</TITLE>

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<DIV>Dennis</DIV>
<DIV> </DIV>
<DIV>I
would say your best bet is to run a 2D SDS-PAGE on your expressed proteins since
their sequence is very similar.  Running a 1D gel would be difficult since,
like you said, their masses are so close.  Having that extra C-terminal
region on one of the proteins should make it separate somewhat easily on a 2D
gel.  After that you could do a western to locate the proteins.  A
second gel could then be run, followed by blotting and some C-terminal
sequencing on both spots to discern one from another.  Another alternative
would be to affinity purify both proteins with your antibody, and then try some
classic chromatography (HPLC, IEX, ect) followed again by C-terminal sequencing
(assuming of course you were able to separate the 2 proteins).  Not sure
which way would be easier. Hope this helps.</DIV>
<DIV> </DIV>
<DIV>Paul</DIV>
<BLOCKQUOTE>
 <DIV align=left class=OutlookMessageHeader dir=ltr>-----Original Message----- From: Dennis Leveson-Gower
 [mailto:dleveson@bccancer.bc.ca] Sent: Tuesday, May 15, 2001 12:25
 PM To: Recipients of ABRF List Subject: Misc -
 Question </DIV>
 Hi everyone,  
 I have a problem and I thought I might be able to
 find some help in this discussion group. 
 I am co-expressing two proteins in CHO cells and
 then trying to detect if one or both of them are expressed.  The problem
 is that the two proteins are identical except for short peptide sequence at
 the C-terminus.  I have antibodies that react with both fragments but not
 with each individually.  The whole proteins should be approximately 50
 and 53 kDa in size so separation on Western would be difficult. 
 
 I need a way to take whole cells, break them open,
 and then see if both proteins are being translated. 
 Thanks, 
 Dennis.
 </BLOCKQUOTE></BODY></HTML>

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