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double total actin and beta-tubulin bands under non-reducing conditions???

double total actin and beta-tubulin bands under non-reducing conditions??? - Western Blot Forum

double total actin and beta-tubulin bands under non-reducing conditions??? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 09-22-2010, 11:57 AM
Pipette Filler
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Exclamation double total actin and beta-tubulin bands under non-reducing conditions???



Hi,

I'm a Biomedical Science honours student. I have a western blot dilemma so I would love any help I can get. I study tetraspanin proteins, in particular CD9. The CD9 antibody I have requires the protein to be non-reduced and the nitrocellulose blots are fine. However, I then bock and stain with either total actin or beta-tubulin III and instead of a single band around 50, i get 2 or three bands around 50!! Does anyone have any ideas as to why this may be occurring? I thought maybe its picking up different isoforms? Therefore it seems i cannot use these as loading controls??
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Old 10-13-2010, 07:10 PM
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Default Re: double total actin and beta-tubulin bands under non-reducing conditions???

Are the double bands in all lanes or only in the outside lanes?
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Old 05-23-2011, 05:21 PM
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Default Re: double total actin and beta-tubulin bands under non-reducing conditions???

I have the same issue. I load lysates from 5 different cell lines and one of them have two actin bands very close together. All lysates are boiled in a beta-mercapto ethanol containing sample buffer and the "doublet" sample is not on the edge of the gel but in the middle.
Any ideas?
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actin , bands , betatubulin , conditions , double , nonreducing , total


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