anti-goat:anti-western blotting??? - Western blotting! (Jun/27/2005 )

Hello everybody!!
Well, I am having problems with antibodies raised in goats, i.e. all which need anti-goat as secondary ab. I get all non- specefic bands but as such background is clear. If I increase blocking, everything dissapears, if I decrease primary or secondary ab, same thing happens. What is the best way of using it?? I am using Santa-cruz abs (for eg Nkx2: sc 8697) and TBS-tween bufferand Amersham's ECL blotting paper and ECl kit..
Hope somebody would give me a good solution!!

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Hi Bluetree,

What are your blocking conditions?

I tend to belive that it is almost impossible to overblock, and therefore could the problem be with the primary antibody, and getting the conditions right there (I'm not familliar with the protein or antibody) although I have had a number of antibodies from companies (incl. SantaCruz) that would not work as well as they should.

Scott

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hi
i get better results with PBS rather than TBS... also using an amersham anti goat

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Thanks you two!!
OK, I'll try PBS and Amersham's, but all other abs from SC work fine except anti-goats.

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