The PI of two proteins are 4.96 and .4.86 and the MW of them are both roughly 15KD. Which concentration of the Bis could the native page need? How about the voltage when running？
Hi there,
It seems difficult to separate these proteins using PAGE... BTW what does roughly mean concerning MW? For proteins exhibiting the same MW and the same pI you need to find a very specific approach (IP or affinity chromatography) to separate them efficiently.
Yes, I agree with Meng Fei. You could follow the principle of Panyim & Chalkley Archives of Biochemistry and Biophysics
Volume 130, 1969, Pages 337-346 They used acetic acid -urea gels to separate histones with isoelectric points in the basic range and low molecular weights. You would need to switch pH to the alkaline range ~9 and use a high acrylamide ~10% concentration. The alkaline pH should maximize the dissociation of the acidic residues. Glutamic and Aspartic acids have a difference in pKa of about 1 pH unit. so you are most likely see differences in charge between your proteins amplified at alkaline pH and the urea should maintain solubility .It might be worth a try. You might look at the Bio Rad pre made gels for something that you could use. A good reference on the effects of various parameters is at the attached link.
http://www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf
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