To make the proteins and protein complexes migrate in PAGE by their mass alone, they all need to be the same charge, normally negative. In SDS-PAGE this is achieved by coating the protein in the anionic surfactant SDS which also denatures structure and complexes.

In Native PAGE, the charge modifier is something like Coomassie Blue (for Blue Native PAGE) or Deoxycholate (for Clear Native PAGE). These are added to sample buffer and often the running buffer. They coat the outside of the proteins and protein complexes giving an overall negative charge but don't denature or disrupt complexes in the majority of cases. So you need to add a charge modifier.

The second thing missing is a mild surfactant in your sample buffer, like dodecylmaltoside (DDM) or digitonin, which assist in keeping the complexes in solution when removed from the native environment.

I would recommend looking up the work of Herman Schagger, especially Nature Protocols 1(1): 418-428 and Proteomics 8(19): 3974-3990.

Dear Veni, to give you  help I need to know why  you are running this kind of gel.

 In my experience you cannot eliminate 0,1% SDS in running buffer because some proteins without own negative charge precipitate in the gel.

First of all I suggest you to run a SDS-PGE and control the migration (MW) of your protein. Then you can reduce in the loading buffer only  the SDS from 3% to 0,1% or alternative you can eliminate only b-Me. In both these case you could detect dimers or multimers . Mobility of basic and acid proteins is not a problem if you know how your protein migrates in SDS-PAGE

good luck    

I agree with Paola and would like to add that the HMW Native Marker Kit from GE Healthcare is a reliable size standard for native protein electrophoresis.

Cheers, Christian

To make the proteins and protein complexes migrate in PAGE by their mass alone, they all need to be the same charge, normally negative. In SDS-PAGE this is achieved by coating the protein in the anionic surfactant SDS which also denatures structure and complexes.

In Native PAGE, the charge modifier is something like Coomassie Blue (for Blue Native PAGE) or Deoxycholate (for Clear Native PAGE). These are added to sample buffer and often the running buffer. They coat the outside of the proteins and protein complexes giving an overall negative charge but don't denature or disrupt complexes in the majority of cases. So you need to add a charge modifier.

The second thing missing is a mild surfactant in your sample buffer, like dodecylmaltoside (DDM) or digitonin, which assist in keeping the complexes in solution when removed from the native environment.

I would recommend looking up the work of Herman Schagger, especially Nature Protocols 1(1): 418-428 and Proteomics 8(19): 3974-3990.

Native electrophoresis can be conducted at different pH-values, depending on the requirements of the experiment. See for example

@ARTICLE{Chr-83,

author = {A. Chrambach and T.M. Jovin},

title = {Selected Buffer Systems for Moving Boundary Electrophoresis on Gels of Various p{H} Values, Presented in a Simplified Manner},

year = {1983},

journal = {Electrophoresis},

volume = {4},

number = {3},

pages = {190-204},

doi = {10.1002/elps.1150040303},

}