Transferring >150 kDA proteins conditions - (Oct/03/2012 )
I generally use a Bio-Rad semi dry transfer machine with 15% methanol and the standard Tris Base/Gycine amounts and I leave it at 0.45mA for 45 minutes for 2 gels, or about 35 minutes for 1 gel and it works very well for most proteins in the range of 40 kDa to up to 100 kDa. Recently, I've been working on a protein that's from 160 kDa to 200 kDa and using that method yields a band at 70 kDa but nothing above that, and weird hollow/ghost lanes show up above 70 kDa.
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.
Wet transfer overnight is usually better in my experience. I usually do 15 V overnight and have no problems detecting p130, I have seen similar results with some trans-membrane proteins such as NKCC1 (190ish kDa).
OK thanks, so semi-dry is definitely not a good bet to try getting larger proteins, regardless of transfer time? Since I've never done wet transfer o/n, are there any potential hazards like over-heating issues even in a 4 degree room?
I usually do my overnights at room temp with 15 V, and the buffer doesn't seem to get any hotter than room temp. You can defintely do them in a cold room if you are worried.
Semi-dry is capable of high MW transfer, but you generally need to optimise the conditions for your protein specifically.