benefits of urea in SDS PAGE gel - (Apr/23/2008 )
hi all!
just a query a lab mate mentioned that putting 8M urea in my samples prior to running a gel may lead to a nicer more resolved gel. is this true? and why if so? and is it normal to run a lower % gel in stacking ie 4% than the resovling gel (11%)?
thanks!
Urea is a strong denaturant. I guess it further denatures proteins and thus they can migrate through the gel more freely. And stacking gel is definitely important to make sure the proteins arrange themselves in such a way that during separation, it can resolve nicely in the resolving gel.
it also helps sharpen the bands.
how does it sharpen the bands? in denatured form? denatured form = sharper bands?
i'm not exactly sure why (maybe because it exposes hidden groups of the protein) but urea enhances charge separations in page. since the protein has a uniform negative charge with sds this effect allows (or forces) the protein to remain stacked.
just a query a lab mate mentioned that putting 8M urea in my samples prior to running a gel may lead to a nicer more resolved gel. is this true? and why if so? and is it normal to run a lower % gel in stacking ie 4% than the resovling gel (11%)?
thanks!
What is the proportion of volume of urea to be added to the sample?
just a query a lab mate mentioned that putting 8M urea in my samples prior to running a gel may lead to a nicer more resolved gel. is this true? and why if so? and is it normal to run a lower % gel in stacking ie 4% than the resovling gel (11%)?
thanks!
As I know, there are two proteins request urea during the extract and the western. One is histone, using acid-urea can get rid of non-histone component from nuclear extract. Another is Insulin, it's small MW molecules and urea are required in the SDS gel. I am not sure about the reason. but the band is always a big ugly patch so I believe to sharping the band is not the reason.
what is the running buffer for insulin, tris-glycine or tris-tricine?
how does the insulin band look without the urea? maybe it is a larger, more diffuse patch without urea?
we have added urea to sds-page to sharpen and improve the separation of high molecular weight proteins. without urea the protein banding was more diffuse and harder to judge mobility.
what is the running buffer for insulin, tris-glycine or tris-tricine?
how does the insulin band look without the urea? maybe it is a larger, more diffuse patch without urea?
we have added urea to sds-page to sharpen and improve the separation of high molecular weight proteins. without urea the protein banding was more diffuse and harder to judge mobility.
We use Tricine-urea-SDS-PAGE system, I agree with you Urea might improve the band, but I won't expect too much.
http://www.jbc.org/cgi/reprint/278/17/14798
http://www.jbc.org/cgi/reprint/278/17/14798
i looked at your reference. the banding doesn't look so much bad as overloaded (either protein load or radioactivity or both). have you tried lower loadings?