[Protein-analysis] "ghost" dots on blot?

Jessica Krakow via proteins%40net.bio.net (by jessica_krakow from targetdiscovery.com)
Tue Jul 17 16:35:53 EST 2007

What causes negative or "ghost" bands/spots on a western or dot blot?

I did a dot blot for determining what dilutions of primary and  
secondary Abs to use.  I spotted about 10 micrograms of total protein  
onto nitrocellulose for each spot and probed with various dilutions  
of my primary and secondary Abs.

I got no signal at all in every spot.  The area where the protein sat  
was much lighter than background.  Too much protein?  Too much Ab?   
Thanks in advance for your help!

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