detect splice variants in living cell

Zhong Silin via methods%40net.bio.net (by stxsz1 from nottingham.ac.uk)
Sun Jul 13 05:37:33 EST 2008


Hi all
 
How can we detect alternative splicing in a living cell? RT-PCR can be used to check alternative splicing. However, mRNA level is not always the same as protein. Promoter:reporter fusion is nearly impossible unless the reporter is in a translational fusion to the target protein. Again, it won't tell the difference of the protein splice variants.
 
I assume that one way is to insert a fluorescent/epitope tag to a specific region of that protein, which is present/absent in its splice variants.
 
Any suggestion please?
 
cheers
silin

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Subject: Methods Digest, Vol 38, Issue 9



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Today's Topics:

   1. RE: IP Problem (Jayakumar, R)


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Message: 1
Date: Fri, 11 Jul 2008 17:41:45 -0400
From: "Jayakumar, R" <R.Jayakumar from roswellpark.org>
Subject: RE: IP Problem
To: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>,
        <methods from magpie.bio.indiana.edu>
Message-ID:
        <97101976F8A044468CA74FE11883B90E173E7ACB from VISTA.roswellpark.org>
Content-Type: text/plain; charset="us-ascii"

Not very difficult at all.  Use a different antibody (for e.g. Mouse)
for IPing the protein down and use another one for western detection
(for e.g. Rabbit).  Then the heavy chain of mouse will not be visible
when we use anti-Rabbit secondary for western detection.

Jay

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Dr Engelbert
Buxbaum
Sent: Friday, July 11, 2008 2:37 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: IP Problem

Am 10.07.2008, 13:43 Uhr, schrieb John Leonard <jleonard.iii from gmail.com>:

> Hello all,
>
> I'm looking to perform an IP Western of a protein that runs at around
> 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's
> heavy chain.  As such, it could be very difficult to detect presence
> of my protein


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