Problems with colony immunoblot

[erickson]

Hi.
	I am trying to perform non-lysing colony immunoblots of yeast, in 
order to detect secretion of CPY (carboxypeptidase Y).
	I have done this several times before with great results. Now, 
however, I am having a tremendous problem with background.  In fact, a 
ran a negative control - just a piece of nitrocellulose, without any 
cells.  I got a very high level of background.
	I just ordered a new ECL detection kit from Amersham - the same 
kit I have always used.  This kit has the detection reagents and the 
secondary antibody.  The nitrocelluose was the same.
	Does anyone have any ideas?  It is frustrating that something I 
have done so well previously suddenly turns sour.  I would appreciate any 
suggestions!  

Louis Geller
Graduate Student - Cell & Molecular Biology
CSU Long Beach.

lgeller at csulb.edu, demode101 at mail.earthlink.net