Reason of Cross-reactions in Western blot? - without specific band (Nov/24/2008 )

I have severe problems with my Cleaved Caspase 3 Western Blot.

I produce protein extracts from kidney tissue using RIPA buffer with protease inhibitor cocktail. loading 20-30µg protein/well.

Can anyone tell me why i got so many secondary bands, but no real specific band?

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if the antibody is specific, you should take less protein per lane; moreover, your blots seem to be overexposed...

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I took only 20µg of protein.
could this be also due to inefficient denaturation of proteins during cooking (e.g. because of false composition of the laemmli buffer)?

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I do not think so; what are you using? minigels? reduce to 15 ug per lane and expose shorter...

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Hi, Try running different amounts of protein on a gel ( like 1 ug, 2 ug, 5 ug, 10 ug.... 20 ug) and do your blot. This will tell you if using a lower protein amount will solve the issue. Avoid overexposing your blot too. Also, try diluting your antibodies further ( too much immunoglobulin can cause cross-reactions with the protein sample).

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do you add tween-20 to your wash and antibody buffers? 0.1-0.2% helps reduce or eliminate non-specific binding.

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