Due to limited sample, I'm trying to maximize each SDS gel/Western Blot that I run. In an ideal world, I'd like to resolve a 40 kD, 65 kD, and 75 kD protein on the same gel.
I prefer to cut my blots into three pieces and incubate each separately with the appropriate antibody (rather than mix all 3 antibodies together). Obviously my concern is between the 65 and 75 kD protein. Fluorescent antibodies are not currently an option for us.
Does anyone have a recommendation for a molecular weight ladder that distinguishes more finely between 50 kD and 75 kD? Does anyone know of a good percent gel that could be used to resolve these proteins? I'm currently using a Tris-Glycine precast gel (4-20%).
I appreciate any tips!
You will want to use a low percentage gel to get the best separation in the 50-75 kDa range. Here is a migration chart for Novex precast Tris-glycine gels. The 8% gel looks the best to me.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/novex-tris-glycine-gels/novex-tris-glycine-gel-performance-data.html?open=migration#migration
You will want to use a low percentage gel to get the best separation in the 50-75 kDa range. Here is a migration chart for Novex precast Tris-glycine gels. The 8% gel looks the best to me.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/novex-tris-glycine-gels/novex-tris-glycine-gel-performance-data.html?open=migration#migration
Dear Samantha,
I agree with Adam. A gel of 8-10 % looks best for me but if you want a better separation of the 65 and 75 kDa proteins I suggest you to use 12% instead and the ladder you can check this http://www.geneaid.com/products/protein/protein-standard-protein-marker-prestained-protein-ladder
Gradient gels can increase resolution as they reduce diffusional band spreading. Use a shallow gradient (say, 2-3% T difference between maximum and minimum). If you add 5% glycerol to the heavy solution, casting the gel becomes easier (it does not influence separation).
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