Before I can answer your question I would like to know more about the kind of native gel (self-made or purchased), any and which detergents, buffer composition and your sample preparation. I have attached my protocol, which may have to be modified for your purposes.

Hi Fatin,

It is usual to observe protein precipitations in the gel wells, for samples which were not solubilized to 100% before electrophoresis.

I would try and use a more efficient solubilization step before electrophoresis.

Best,

Hediye.

Hediye Erdjument-Bromage this issue only happens when I run native PAGE. It never happened in reducing condition. why is that so?

You may need to experiment with the detergent and/or the concentration of detergent to facilitate migration of the proteins into the gel. Another possiblity could be that there is too much acrylamaide/bisacrylamide and your proteins can't enter the gel.

Native gels requie that you do not denature your protein. So, it is not surprising that you are having issues..

There needs to be a mild detergent to facilitate the solubilization of your protein. The fact that the issue does not happen when you use reducing conditions, (disruption of the S-S bonds) indirectly confirms that unfolding/solubilization of starting protein material prevents the precipitation of the sample in the gel well.

Here is an excellent review by Ilka Wittig and colleagues:

Chapter Mitochondrial Complexome Profiling

which we find very detailed and useful in our laboratory.

Best,

Hediye.