Cross banding on Westerns using monoclonal Ab? help!

Mr. A.F. Hill ahill at crc.ac.uk
Tue Jun 1 11:19:45 EST 1993


hi there

i am trying to perform some western blots on some proteins extracted from
E.coli which contains an expression vector for a protein we wish to study
(ie to use as an assay for expression). i am using a monoclonal antibody for
the primary and either a horseradish-peroxidase conjugated secondary antibody
(use ECL to develop) or a alkaline phosphatase conjugated secondary antibody
(NBT & BCIP to develop).

the problem is that several bands develop on the membrane - including bands
from the molecular weight standards that were loaded onto the SDS-PAGE gel.

is there any reason why several bands appear - obviously unrelated to the
protein of interest, and which shouldn't (i presume) react with the monoclonal
primary antibody?

any help would be appreciated...

Andy

==============================================================================
 Andy Hill				  |		
 Prion Disease Group			  |     E-Mail: ahill at uk.ac.crc	
 Dept. Biochemistry & Molecular Genetics  |-----------------------------------  
 St Mary's Hospital Medical School	  |     Phone: 071 723 1252 ext. 5469
 LONDON W2 1PG				  |     Fax:   071 706 3272
==============================================================================



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