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+--- Thread: Problems with Western Blotting!!! (/thread-190.html)
Problems with Western Blotting!!! - labvamp11 - 08-08-2011
Western blotting…*sigh*…I have been performing Western blots on my peptides which are labelled with both a GST tag and a HIS tag on their N- and C-terminal ends respectively. Whenever I perform the blot with an anti-GST antibody, it works perfectly, yet whenever I perform the blot with the anti-HIS antibody, it never works properly. My samples would never appear, and my controls would be faint. I cleaved the GST tag from my peptides (with Thrombin, stopping the reaction with PMSF) and then performed a blot with the anti-HIS antibody again, and now nothing shows up, only non-specific blotches all over the exposed film!
If anyone has any suggestions as to what might be interfering with it, I’d greatly appreciate it. Thanks
FYI, I have also posted this discussion on Nature Network.
http://network.nature.com/groups/labvamp/forum/topics