I'm going to do native page and i just want to know its principle completely.
The principle for native gels when Coomassie is not added to the sample is that proteins are separated by a combination of size and charge. The charge in general depends on the number of amino acid residues that bear a positive or negative charge at the pH of the gel. So if running the gel at pH from 8.3 / 8.9, Asp and Glu will be negatively charged, Lys and Arg and His will be protonated and have a positive charge. The N- terminus would have a positive charge while the C terminus would have a negative charge. There might be exceptions depending upon the micro environment of each residue. Native gels can be run at acidic pH as well, to give another way of resolving proteins. Smaller proteins migrate faster than larger proteins. Also, quaternary structure is preserved, so a dimer will run as a dimer so the percentage of total acrylamide monomer used is usually lower than what is used for SDS-PAGE, e.g. 7.5% acrylamide.
the anionic dye Coomassie blue G-250 give net charge! and the separation is generally based on the size of the molecule.
please refer the attachment
In blue native gels, proteins are charged by using high quantities of Coomassie Blue G-250, both in the sample and in the gel. Binding a large number of dye molecules imposes a charge shift on the proteins that causes even basic proteins to migrate to the anode at pH 7.5.
coomassie gives charge to the protein in Blue Native PAGE... but in normal Native Page protein migrates driven by its own charge...
And you can try to calculate this charge from pI of the protein and pH of your buffer for Native PAGE without Coomassie
The principle for native gels when Coomassie is not added to the sample is that proteins are separated by a combination of size and charge. The charge in general depends on the number of amino acid residues that bear a positive or negative charge at the pH of the gel. So if running the gel at pH from 8.3 / 8.9, Asp and Glu will be negatively charged, Lys and Arg and His will be protonated and have a positive charge. The N- terminus would have a positive charge while the C terminus would have a negative charge. There might be exceptions depending upon the micro environment of each residue. Native gels can be run at acidic pH as well, to give another way of resolving proteins. Smaller proteins migrate faster than larger proteins. Also, quaternary structure is preserved, so a dimer will run as a dimer so the percentage of total acrylamide monomer used is usually lower than what is used for SDS-PAGE, e.g. 7.5% acrylamide.
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