Transferring >150 kDA proteins conditions
Posted 03 October 2012 - 12:20 PM
I changed the transfer buffer to 10% methanol, with 0.1% SDS and the same Tris Base/Glycine amounts and transferred at 0.2mA for 90 mins but now I see no band. I realize that using semi-dry to transfer large proteins might not work as well but was wondering if anyone has succesfully done so and under what conditions?
A colleague lent me a wet transfer system but set the conditions to 100V for 60 minutes. I thought that the slower the better for large proteins, so would 75V for 4 hours be better or 30V for overnight? The protein was run on 7.5% gel.
Posted 03 October 2012 - 01:09 PM
Posted 03 October 2012 - 02:43 PM
Posted 04 October 2012 - 12:12 AM
Semi-dry is capable of high MW transfer, but you generally need to optimise the conditions for your protein specifically.