The objective of pre-equilibration is to re-nature the SDS-PAGE resolved proteins, as much as possible; so that when transferred to nitrocellulose (or other electroblotting material) the proteins have regained their tertiary structure which may be important for antibody binding or intrinsic enzymic activity. Methanol is needed to remove SDS from the proteins in the gel prior to and during, transfer so that renaturation can occur.
Hello Tejas,
I use to do it, but I wet the gel in the cold transfer buffer only ten seconds, before I lay it on the PDVF membrane. This way, there's no problem with the gel.
The objective of pre-equilibration is to re-nature the SDS-PAGE resolved proteins, as much as possible; so that when transferred to nitrocellulose (or other electroblotting material) the proteins have regained their tertiary structure which may be important for antibody binding or intrinsic enzymic activity. Methanol is needed to remove SDS from the proteins in the gel prior to and during, transfer so that renaturation can occur.
Hi,
Like already mentioned the purpose is to get back the re-naturing of your protein for better transfer on the membrane and hence detection through Ab. I pre-equilibrate for a few minutes. Whether you use PVDF or Nitrocellulose, gels have to be in cold-transfer buffer with methanol.
Hi, as people mentioned above, but if you let it too much you may lose signals as proteins may diffuse out of the gel.
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