Protein does not migrate into resolving gel

[lindberd]

I am currently trying to investigate an enzyme secreted from an unknown marine bacteria. I have collected supernatant from bacterial shake flask cultures and aim to analyze these supernatants via SDS-PAGE to identify the size of the enzyme and possibly use MS to sequence the enzyme.

Problem is that the protein is not migrating into the resolving gel. Both my protein marker (Fermentas Prest. Prot.) and the dye front of the sample buffer seems to migrate properly, but when the gel is stained with Coomassie, the only band I get is an extremely smeary one at the stacking/resolving gel interface. It seems like the sample has "stuck" at the resolving gel and then just diffused throughout the stacking gel.

I use BIO-RAD precast Mini Protean TGX gels of 4-20% and a MOPS running buffer. Running gels at 180V for ca 40 minutes. Samples are boiled prior to loading and the loading buffer contains appropriate amounts of BME.

Any ideas?

[obama]

Hi lindberd,

Add some more points like:

How you processed the supernatant: Which buffer you dissolved it, did you boiled it before loading. Did you measured the protein conecntration if yes then how much you loaded.

Then one can get an idea what's going wrong.

[Christinemfz]

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