IP Problem

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Fri Jul 11 13:36:31 EST 2008


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

This largely deppends on how you want to detect your protein after  
electrophoresis. Usually in IP you have too small an amount to detect by  
protein staining. Instead the target protein is usually metabolically  
labelled (originates from cell cultures grown with 35-S Met/Cys), the  
presence of Ig heavy chain would not interfere with detection as it is  
non-radioactive.

A little technical hint: Protein A beads are quite expensive. For IP I use  
fixed Staph. aureus cells instead. These are commercially availble (e.g.  
Pansorbin from Calbiochem).


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