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Separating close bands in Western Blot

western blot Tris-Glycine acrylamide resolution

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3 replies to this topic

#1 msc10

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Posted 02 October 2011 - 02:48 AM

Hello,

I am having trouble trying to separate with a Tris Glycine WB two proteins with a very similar MW: 65 and 63 kDA. Is there any program to calculate the best % of acrylamide to obtain the best resolution in this weight range? Do you have any tips to get sharper bands and avoid overlapping of those so close bands?

Thank you very much in advance!

#2 Papaver

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Posted 04 October 2011 - 03:09 AM

Maybe a gradient gel might help.

#3 JDiamond

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Posted 04 October 2011 - 08:09 AM

I would try a 10% gel and be sure to run the dye-front off the bottom of the gel. You may want to continue to run the gel even longer to ensure greater separation of your proteins. -Julie Diamond, Cell Signaling Technology Product Scientist

#4 mdfenko

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Posted 05 October 2011 - 06:37 AM

separation of 2 kDa in that size range will be very difficult.

you should limit your loading (volume as well as mass) to no more than can remain sharp after stacking.

you can improve band sharpness by adding urea to the gel (up to 8M).

you may want to try a different separation technique (eg reverse phase, ion exchange or hydrophilic interaction hplc) depending on the characteristics of the proteins.
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