Dear colleagues,
I am currently using gradient Tricine-Imidazole native PAGE supplemented with deoxycholate (the so-called "high resolution clear native PAGE") in order to analyze the native oligomer state of my (membrane-associated) proteins of interest. The overall separation is very good and I do not face any problems with thansfer and detection of high molecular weight proteins (100-600 kDa).
A brief trobuleshooting history: time of transfer varied (0.5 h - 5 h) - no much difference in tranfer effiency. I was aware of over-transfer of low molecular weight species, so I tried to blot to double layer of PVDF (however, the proteins of interest were not detected as well).
Any ideas will help.
Best,
Vladimir
Hi Vladimir: the Deoxycholate-native PAGE separates proteins not based on its sizes along but both sizes and charges. So, your 30-70 kDa proteins may not necessarily migrate at positions seen on SDS-PAGE. A key issue is to wash away any residual deoxycholate in the gel before transferring to PVDF (in transfer buffer for at least 30 min), to increase transfer efficiency. Good luck.
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