pinkc

This was run on 7% gel, transferred at 100v for 1 hour.  I've never done wet transfer before, but I can tell that the really black borders around the lanes are abnormal.  Is it because of protein overloading?  In addition, the band that's shown is actually at 70 kDa and the protein of interest should be 160 kDa - 200 kDa.  I've tried semi-dry transfer without success so I guess I'll run less protein and wet transfer for overnight.  Any suggestions would be welcomed, thanks!

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.

I am following a protocol that requires the stacking buffer be adjusted to a pH of 6.8.  I accidentally overadjusted it to around 6.1 so I used some NaOH to compensate the pH back to 6.8.  Would this affect the stacking gel performance or not really?