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Thank you very much, I will send you a scanned blot with RIPA lysate tomorrow, I haven't tried that. Althgouh I am not sure whether that will answer my question if the higher molecular weight band is specific for LIm1 or not. The samples were boiled 5 minutes in sample buffer. Thanks, I hope you can help me further,
Asked on Sep 08 2011
Thanks for your reply and I look forward to receiving your image. Regarding the 150 kDa band, I have looked through our records and briefly in the literature and have found no evidence that LIM1 should run that high, so this band is most likely non-specific. Using RIPA buffer will ensure that all of the LIM1 in the nucleus is released, which may increase the strength of the specific band but won't affect the non-specific band. Also, using RIPA (contains SDS) and boiling the sample in reducing and denaturing buffer (like Laemmli) will ensure that the protein is running as a monomer and should be at the expected weight. Please let me know if you have any questions. We do guarantee all of our antibodies to work as stated on the datasheet in tested species and applications, so I'll be happy to provide a replacement, credit, or refund if the antibody is not working as it should be.
Answered on Sep 08 2011