Western DotBlot protocol?

Colin Rasmussen crasmussen at anat.med.ualberta.ca
Tue Apr 25 00:54:02 EST 1995


blume at tigger.jvnc.net (John E. Blume) wrote:
>Does anyone have a favorite protocol for doing Western blots in some 
kind
>of dot/slot format?
>
>I have many samples to test for effect and I hope to avoid running a 
gel
>and transferring. Obviously, I am concerned about background and about
>membrane binding capacity, but it strikes me that someone must have 
this
>worked out.
>
Offhand I can't remember the precise protein binding capacity of typical 
membranes, but we have used nitrocellulose and Immobilon-P with great 
success.  We usually resuspend the protein of interest in either Milli-Q 
water or PBS and apply to the filter.  After dot/slot-blotting we then 
proceed directly to the detection protocol.  If you are worried about 
overloading the filter, the simplest thing to do is apply a series of 
serial dilutions.  You will eventually find a linear response.





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