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SDS Sample Buffer
Posted by: EANON (IP Hidden, Junior member, 19)
Date: July 31, 2005 03:32PM
I am running a tagged protein on an SDS-PAGE. When I immunoblot for the tag, I see the strong band (and nothing in the control). However, this appears at a much higher MW than expected. I suspect my protein is migrating as a complex.
Does anybody what ingredient I can add to the SDS sample buffer or modification to the protocol to separate the protein from the complex?
Re: SDS Sample Buffer
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: July 31, 2005 05:20PM
If you were running non-denaturing native gel and want to change to denaturing SDS-PAGE to disrupt any protein complexes. Here is the sample buffer you can use:
SDS 0.5g beta-Mercaptoethanol 0.25ml in 50ml Tris-HCl pH 6.8 buffer and add bromophenol blue. Edited 1 times. Last edit at 07/31/05 05:44PM by mitolab.
Re: SDS Sample Buffer
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 1, 2005 09:30AM
You may also try boiling the samples for 3-5 minutes before loading them into the gel. this will also help denature the proteins.
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