Hello everyone. I want to do a Native-PAGE followed by a western blot analysis to detect the different native forms of my proteins of interest (synuclein family). Could anyone have a protocol or some advice to give me? For example, related to the lysis buffer that I have to use and the running buffer. Is it necessary to run the gel in a cold room? Any tip would be useful. Thanks you!
You can try regular Tris glycine gels à la Lammli, but leasing out SDS and mercaptoethanol from all buffets. Don’t forget that the migration will give no indication on the size of the species unless you perform Ferguson analysis with gels containing different concentrations of polyacrylamide.
You can try regular Tris glycine gels à la Lammli, but leasing out SDS and mercaptoethanol from all buffets. Don’t forget that the migration will give no indication on the size of the species unless you perform Ferguson analysis with gels containing different concentrations of polyacrylamide.
Hi Florencia Malizia,
I totally agree with Dr. Pierre Béguin.
I've done a lot of Native-PAGE and I used the same protocol as SDS-PAGE without any denatured-agents: no SDS and Mercaptoethanol/DTT in sample buffer, no heat treatment, and you don't need to run the gel in a cold room (Unless your electrophoresis unit heats up a lot during the operation and your proteins are heat sensitive).
Just notice that the non-denatured protein could be different from the denatured protein in structure, so you might need to check back and try optimizing the suitable time for the transferring step in western blotting.
Best luck!
I think a popular buffer system is a 1X running buffer solution:
25 mM Tris-HCl (pH = 8.3), 192 mM Glycine, 0.003 % m/v Coomassie G-250.
Use of Coomassie G-250 allows you to do Blue Native PAGE. Coomassie G-250 gives proteins a negative charge, while still maintaining the native structure of the protein.
As others have said no SDS in the gel or in the running buffer.
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