Hi everyone,

we are trying to establish BN-gels in our lab and we are currently facing some problems. We would like to investigate protein-protein interaction of membrane bound proteins from cell lysates.

In our setup we are using precast TGX gels from BioRad (without SDS), our running buffers are based on Tris/Glycine like the gels and supplemented with 0.02 or 0.002% CBB-G250. The lysis buffer we are using is composed of 62.5 mM Tris, 12 mM NaCl, 250 mM aminocapronic acid, 250 mM sucrose, 10% glycerol, 1% Triton X-100, pH 7.0. Before loading, we add 0.5% CBB-G250 (from 5% stock in water) to our samples.

The gels are run in a cold room at 100 V, after 15 min the dark blue running buffer is switched to light blue or clear one.

We can see, that the samples enter the gel and continue running through until they reach the last third. But there is always this dark blue smear at the running front, which at some point does not migrate any further in the gel (see in the photo), even when the gel runs for >20 h. There are also no protein signals detectable beyond this point. We have already tried different concentrations of aminocapronic acid, sucrose and Triton in the lysis buffer, but we were not able to fix the problem yet.

Does anyone have suggestions for us, what we could do to fix this problem? I am thankful for any advice.