Tris-Tricine SDS-PAGE problems - (Jun/18/2007 )

Hi all. I need advice to sort out the problems I have with Tris-Tricine PAGE of insulin after limited digestion.

First of all, the bands of both undigested and digested protein have a large downward smear although the protease band is perfectly sharp.

Second, I get only one band even with freshly prepared DTT-containing sample buffer (I use pH 6.8 Tris-HCl, 8% SDS in my sample buffer, 12% running gel with glycerol). Partially reduced band is lower than non-reduced but it's above the insulin B-chain marker that's why I think it's due to incomplete reduction even in digested samples.

And finally, at least with reduced samples, when I apply less of the same sample, the band (it looks more like a patch because of the smear) ends up way lower. NaCl final concentration in the sample is 25 mM.

I would appreciate your suggestions. Thanks a lot in advance.

are you boiling your samples before applying to the gel?
are you using a stacking gel? spacer gel?
could you use a gradient gel (10-16.5% or 10-20%)?
if you are not resolving two close bands then try higher %age gel (16.5%). a steep gradient might also work.