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Immunolotting of actin protein coming at higher MW


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#1 repeatcell

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Posted 18 June 2009 - 10:01 AM

Hi

I prepared my protein samples in SDS sample buffer and ran a gel using Invitrogen pre-casted gel and used a protein marker (pre-stained) on a PVDF membrane. When I did immunoblotting of my samples, I now get an actin band at 55 KDa with one antibody (Neomarkers) and at 82 KDa with another antibody (Sigma) which is of course higher than the normal mol. wt of 42KDa for actin. Both the antibodies are doing well with samples of other people. Any ideas what I am doing wrong??

Thank you in advance for your help!!

Edited by repeatcell, 18 June 2009 - 10:13 AM.


#2 mdfenko

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Posted 19 June 2009 - 06:56 AM

are you heating the sample after adding the sample buffer? if not you may be seeing a dimer and an improperly unfolded protein (although 55kDa is in the range of the keratin artifacts).
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