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Bromophenol blue vs. "Coomassie G250"


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#1 MDavies

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Posted 09 November 2009 - 08:37 AM

I have a simple question -- We are trying to detect a very small protein, which should be around 18 kDa although some people say it's detected around 14 kDa.

We normally cut off the dyefront after the proteins run through the gel. (Bis-Tris, 10%, using the NuPage system)

Does it really make a difference to change from using bromophenol blue in the sample buffer to using the NuPage buffer with Coomassie G250 in it? It says that will create a dyefront that runs closer to the ion front than bromophenol blue...thus some smaller proteins/peptides which are cut off with bromophenol blue will not be cut off with G250.

But I can't find out what size of peptide this would make a difference for. Does anyone know exactly where, in terms of "kDa", these two dyefronts normally run?

(also, I assume G250 is some sort of special formulation of Coomassie that doesn't stain proteins...)

Thanks!

#2 mdfenko

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Posted 09 November 2009 - 09:10 AM

(also, I assume G250 is some sort of special formulation of Coomassie that doesn't stain proteins...)


no, g250 will stain proteins. it is more specific than r250 so it will stain less. it is also used in the bradford protein assay.

i'm a little surprised that someone suggests using it as a tracking dye for proteins. i would use phenol red if i wanted it to run faster than bromphenol blue.
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#3 lab rat

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Posted 09 November 2009 - 09:14 AM

Hi MDavies,

The difference between G250 and R250 is the color (green- vs red-blue) and one gives a better-resolved band than the other. (I don't remember which one.) Both stain proteins.

What is the smallest band on your MW marker? Have you tried using a low-range marker? I use Amersham RPN800 full-range dual-view, which has a 12kDa band. I have also left the NuPage dye front on my gel and had no problems with finding small proteins.

regards,

lab rat
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.




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