blocking in northern blots

Lauryl Maiya J Nutter lmjnutte at acs5.acs.ucalgary.ca
Fri Feb 2 19:39:30 EST 1996


Doug Craig (doug at hobbes.chem.ualberta.ca) wrote:
: I am trying to stick target DNA to a piece of nitrocellulose, bind 
: biotinylated probe to it and detect with streptavidin-alkaline 
: phosphatase conjugate. I get a high background. Does anyone out there 
: have a favorite blocking method that works well? So far I have been 
: blocking with herring DNA and BSA. I am using a very high sensitivity 
: alkaline phosphatase assay which I have developed and require very 
: effective blocking.

Doug,

For all of our purposes (Southern and Northern blotting) using
conjugated alkaline phosphatase detection methods, we use a 1%
solution (w/v) of Boehringer Mannheim blocking reagent (cat.
no. 1096 176) in 100 mM maleic acid, 150 mM NaCl, pH 7.5.  This
blocking reagent is purified proteolytic fragments of casein and
I have been told that skim milk powder will work, but the
concentration needs to be increased since there are other things
besides protein in milk powder.  

Standard disclaimers about not being associated with BM, etc.

Hope this helps.

Good luck!

Lauryl Nutter
University of Calgary
lmjnutte at acs.ucalgary.ca



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