Hi,
One of my protein is 100kDa on SDS PAGE gel. I have a strong hunch that it forms trimer in the native conditions. It is eluting at correct trimer position in Sephadex 200 10/300 GL column. I want to estimate the exact molecular weight of the protein. It is an acidic protein with 5.9 pI.
What should I use colourless native PAGE or Blue native page? Moreover, can I use routine SDS PAGE markers available to determine the molecular weight in native page??
A single native PAGE gel cannot tell you the molecular weight of the naive protein because migration is also affected by shape and charge of the protein. To use native PAGE to measure the molecular weight, you must run several gels of different acrylamide percentages. This is explained here:
https://www.nationaldiagnostics.com/electrophoresis/article/native-protein-electrophoresis
Other methods of measuring the native molecular weight of oligomeric proteins include dynamic light scattering (DLS), size exclusion chromatography combined with multiangle light scattering (SEC-MALS), and analytical ultracentrifugation. You can also use SDS-PAGE combined with mild chemical crosslinking.
Thanks a lot. Adam B Shapiro . What about the protein ladders to be used?
They are commercially available, e.g.
https://www.thermofisher.com/order/catalog/product/LC0725
or you can make your own using whatever pure proteins you have in your lab for which the molecular weight is already known. If you do that, you should run them individually in separate lanes in order to known which is which, since they won't necessarily run in order of molecular weight.
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