immunoblotting

Jean-Pierre Szikora szikora at licr.ucl.ac.be
Fri Feb 12 11:55:24 EST 1993


>> a simple experiment, but not working...
>> who can help ???
>> I did immunoblots (running ag on a gel, blotted it, incubated blot with
>>		   positive human serum, developed with anti human POD
>>		   conjugate  ==> no bands !!)
>> Ponceau staining revealed lots of protein on my blot.
>> My substrate reaction is positive, too.
>> After several investigations the problem is bordered in:
>> 	no binding of the antibodies (or not enough) in the positive
>> 	patient serum to the on NC membrane presented antigens !!
>> why ?? did anybody experience  similar problems ??
>> in addition it must be said, that i did the same procedure as ELISA (same
>> antigens coated, same human serum, same POD conjugate !!) - and there it was
>> working very well !!!

>> So any ideas what prevents ab binding to NC presented antigens ????
>> I appreciate every hint, thougt or idea. Thanks.

hello,

maybe the reason why you observed differences
between NC et elisa experimentations is that
your positive serum contains antibodies against specific
conformational epitopes.
That's why it work's well with elisa (where antigen integrity is
preserved) and not after NC blotting (where antigen is "linearized").
One solution: test other patients sera containing (?) ab.against sequential
			  epitopes  .

good luck.

Michel Somville PHD.
Ludwig Institute for Cancer Research 
Brussels Branch.

E-mail: Somville at LICR.UCL.ac.be  
Snail:02/764.74.76
Fax:02/762.94.05
 



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