Hi Akshay

I suggest you try simple agarose gels. If you locate the comb in the middle of the gel, as it is often done when you want to run a lot of DNA samples, you will see proteins running in either direction. The only difference is that the straining/destaining will be solower.

Two possibilities:

1. Your protein precipitated on the top of the gel.

Because your treatment buffer doesn't contain detergent, it is very likely to happen

2. Because the protein does not move at all, it doesn't have charge. You need to change your buffer to make the protein negatively charged.

Understanding your issue, i will suggest to purify these using SEC preferably on an FPLC.

@cydi .. dont you think that adding buffer in native gel will denature the protein and it will be similar to SDS PAGE?

Second point I am using my buffer and gel pH > pI so protein have negative charge.

You can use non-denaturing detergent, such as glucoside, even Triton-100.

How do you know the PI? Calculated PI is based on sequence. It is apparent PI. For a folded protein, some charge residues are buried inside, their contribution to PI are hard to be implemented into calculation. As a result, the actual pI can be different from apparent pI