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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Western Blot
Posted by: Guilherme (IP Hidden, Unregistered user, )
Date: April 14, 2005 02:49PM
Hello everyone,
I am doing western blot using a recombinant protein. I transfer the protein to nitrocellulose membrane and confirmed by Ponceau-S staining. The secondary antibody is HRP mouse anti-human IgG1 (1:5000).I donīt know why my secondary antibody, reconignize all the protein. I am thinking to use the HRP goat anti-human IgG1, because when I use goat anti-human IgG my western blot works. What do you think about this? Thanks in advance, Guilherme
Re: Western Blot
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 14, 2005 03:36PM
Guilherme,
I am not sure, but it sounds as if your problem is that the secondary antibody is detecting every protein on the membrane resulting in multiple bands of varying molecular weights. Too many bands being recognized can occur for several reasons including: protein overloading (highly concentrated immobilized proteins may allow for non-specific binding of certain IgGs); proteolysis of the antigen (multiple bands of lower molecular weights than the full protein will be seen), insufficent blocking; antigen concentration too low (relative concentration of the protein of interest not high enough to be readily detectable, subsquent signal enhancement such as ECL may then lead to the appearance of false bands). I believe there is a troubleshooting guide for this problem listed on this website that may give you a better understanding. If I remember correctly it is listed as "too many bands on a western blot". I think you might want to make the switch to HRP goat anti-human IgG1, after all whatever works, works. Plus it is possible that non-specific binding is less likely with the goat anti-human IgG1 than with the other IgG, since you do not see too many bands. Hope this helps! :)
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