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Why immunoblotting of actin coming at higher Molec. Weight


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#1 repeatcell

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Posted 18 June 2009 - 12:11 PM

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 - 12:17 PM.


#2 Nrelo

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Posted 21 June 2009 - 07:02 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!!


Pre-stained markers are only for rough estimation of M.W. The dye conjugated to the protein can affect their migartion. Their migration can be very different in gels with different compositions or % acrylamide (migration of your proteins is also a matter). If you have a positive control in the gel, stick to that control

I got experience on this: I used pre-stained markers from two brands. A 65kDa band from brand A was at the same position of a 140kDa band from brand B in 10% gel...

Edited by Nrelo, 21 June 2009 - 07:03 AM.





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