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Denaturing protein before blotting


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#1 mcb56

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Posted 19 October 2010 - 01:26 PM

I have a general question about denaturing the protein before blotting. I know it is typical practice to run the protein on a denaturing gel with SDS loading dye prior to transfer. If the protein is denatured on the blot, then how can the antibody recognize it? The protein is unraveled and the antibody is designed to recognize the properly folded version.

Thank you

#2 rkay447

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Posted 19 October 2010 - 01:53 PM

This is why some antibodies do not work for western blotting. There are two different types of epitopes an antibody will potentially recognize, conformational and linear. If the epitope is conformational which means it recognizes a specific 3D structure, the antibody will not work in western blots. First, this is why some people generate antibodies to a very small peptides. This is more likely to generate antibodies that recognize the linear epitope. Also, this is one of the benefits of rabbit polyclonal antibodies that consist of a wide variety of antibodies that recognize many different epitopes within the target protein but also increases the chances for background/non-specific binding problems. You never know in a rabbit polyclonal antibody how many of the antibodies are not recognizing the denatured protein but as long as enough of them are binding the specific protein, you've got yourself a good antibody for western blots.




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