I want to determine if my protein forms a dimer in certain conditions. I run the native page to determine it. all the buffers are same to SDS page buffer except for SDS, DTT, beta-me. then i transfer it directly to membrane. The result seems there is no dimer formation. But since I have never run it before, I am not sure if it is right. And other proteins in same family are easy to form dimers.
So should I judge it? And if there are some details or protocols for native page. My lab has no sufficient materials for blue native page.
thank you
You can also try performing a gel filtration experiment to measure the molecular weight of the protein, using protein standards to calibrate the column. Another method that you can try is chemical crosslinking + SDS-PAGE.
If the apparatus is available, other powerful techniques are analytical ultracentrifugation, and size exclusion chromatography with multiangle light scattering. Another method requiring special equipment is dynamic light scattering.
You can also try performing a gel filtration experiment to measure the molecular weight of the protein, using protein standards to calibrate the column. Another method that you can try is chemical crosslinking + SDS-PAGE.
If the apparatus is available, other powerful techniques are analytical ultracentrifugation, and size exclusion chromatography with multiangle light scattering. Another method requiring special equipment is dynamic light scattering.
You can used HPLC with TSKgel column on light-scattering detector and your required dialysing buffer will be your mobile phase.
Use gel filteration Sephadex G200 column for separation of protein under native and then under reducing-denaturing condition using SDS/DTT. In each case you need to calibrate a reference profile between elution volume and corresponding A280 value using a mix of three proteins of known molecular sizes.
Alternatives would be analytical ultracentrifugation (doi:10.1110/ps.0207702) or light scattering.
If you run your gel filtration column with SD, you can ditch the resin after the run. You will never get rid again of all the SDS bound to the resin matrix.
Achim Recktenwald has touched an important point. Another aspect of this is that the first equilibration of the column with the detergent-containing running buffer takes several days, due to the micellar nature of detergents. The buffer can be pumped over the column slowly in a closed loop, that minimises the need for supervision. Only after this step will you get reliable results from your column.
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