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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Stripping buffer
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: August 12, 2005 03:09PM
Can anyone here recommand a good stripping buffer? Thanks.
Re: Stripping buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 15, 2005 03:47PM
Stripping buffers use either pH, heat or detergent to remove antibody. The most common stripping buffer (100mM Mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl buffer at pH 6.7) uses heat and detergent. To use this one: submerge the membrane at 50C for 30 minutes with gentle agitation. An alternative to this is Glycine stripping buffer (0.2M Glycine, 0.05% Tween-20, pH 2.5) which uses an acidic pH to remove the antibody. One advantage to this buffer is that it lacks mercapthoethanol, hence no bad smell. To use this one: submerge membrane at room temperature for 15-45 minutes. You may want to test for remaining antibody after every 15 minutes with this one. Prolonged use can strip protein as well as antibody.
Re: Stripping buffer
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: August 16, 2005 08:54PM
femeauburn, thanks a lot. I'm going to try the Gly with Tween 20 one next week. By the way, the technician in our lab used the one with Mercaptoethanol and found that her antigen proteins were also stripped out.
Re: Stripping buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 17, 2005 11:50AM
nice to know it didn't just happen to me. :)
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