Western Blotting - Electrophoretic transfer of proteins (Apr/26/2005 )
I would like to know what is the best choice to perform electrophoretic transfer of my proteins (20-200kDa) to a PVDF membrane? At a constant voltage (V) or at a constant current (A)? Could anyone explain me the difference between using one or another? I would like to know the conditions commonly used.
Thank you very much.
I use a constant current and always transfer in tris glycine buffer (6.06g trizma base, 28.8g glycine, 400ml MeOH, 1600ml ddH2O = 2 litres)
I use a constant current for my transfers usually 300mA for 90 minutes. The buffer I use is really simple and works very well- 22g CAPS, 1L MeOH and bring up to 10L with distilled water. Needs to be at pH 11.0 which is achieved by addition of 10N NaOH