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Ghost band in SDS-PAGE........why?!?!?!?!?!?
Posted by: Mari (IP Hidden, New member, 2)
Date: March 21, 2006 06:21AM
I’m running an SDS-PAGE (17%) on a purified protein.
I can see clearly the band at the right molecular weight of my protein (15 KDa) but I see also a band at the double value (30 KDa). I used DTT in the sample buffer so It can not be due to a disulfide bridge. So my question is: how can I know if that band at 30KDa is real or simply a ghost band? And if is it a ghost band, how can I remove it? Thanks in advantage! Marica.
Re: Ghost band in SDS-PAGE........why?!?!?!?!?!?
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: March 26, 2006 09:53AM
Take a look at this paper:
Ochs, D. (1983). Protein Contaminants of Sodium Dodecyl Sulfate -Polyacrylamide Gels. Analytical Biochem. 135: 470--474. It is possible keratin cross-react with your protein. But the MW of keratin is > 40KD. You still can try to avoid keratin contamination by wearing gloves, using new bottle of b-mercaptoethanol and see if the ghost band disapper.
Re: Ghost band in SDS-PAGE........why?!?!?!?!?!?
Posted by: StanleyCheung (IP Hidden, New member, 1)
Date: April 9, 2006 11:44AM
How did you purify the protein? Did you boil and centrifuge your samples before loading?
Re: Ghost band in SDS-PAGE........why?!?!?!?!?!?
Posted by: Rosalind324 (IP Hidden, New member, 9)
Date: May 17, 2006 12:11AM
Try western bloting(WB) if possible
just to confirm if the ghost band are protein's aggregation (maybe too many s-s bonds lead to several protein aggregation state
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