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},

}

Matthew P Padula what percentage of e.g. Coomassie would you recommend to add to the sample buffer and running buffer?

Julia Caspers To prepare 2x sample loading buffer you need 200mM Tris HCl pH8.8, 20% Glycerol, and 0.005% (w/v) bromophenol blue. To prepare bromophenol I suggest to make 0.1% stock. And as for running buffer 0.002% for light blue cathode running buffer. I hope it helps you. good luck

Pedram Yadollahi thank you for your answer! I actually used the sample buffer you mentioned but my samples did not move at all. Afterwards I tried adding coomassie to my sample buffer and adding deoxycholate to my running buffer, without any movement of my samples.

Are you telling me to use this sample buffer and add bromophenol blue to my running buffer?

Julia Caspers I used bromophenol blue and added 5% G250 (#BN2004, Invitrogen) it is similar to have coomassie in sample buffer. you can try two buffer system. you prepare your running buffer as usual but your inner reservoir should have light blue or dark blue running buffer. I had problem with movement of my samples after trying this method it worked. To prepare dark blue and light blue I used 20X Cathode buffer (#BN2002, invitrogen). note i used this in biorad system.

Great, I am also using the biorad system. Thank you a lot

Pedram Yadollahi, I will try out your method!

Julia Caspers cool let me know if it will work for you too. Good luck

I wonder, why you're not heating it? I heat for 5 mins at 90 degrees Celsius and I am using 4X loading dye instead of 2X, which will end up being lesser volume (ul) of the sample that one would load on gel. Hence, less spillage or carry over to adjacent well. I run at 120 V for 10 mins, once samples enter resolving gel, I lower the volts to 120 V.

Good luck!

Pedram Yadollahi unfortunately your suggestion did not work out for my samples and Standard Proteins.

Julia Caspers I am sorry to hear that your protein sample is whole lysate or pulled down protein ? if it is pulled down you may want to check your isoelectric point (PI) of your protein. if you could upload some image it could help more. To see how your gel looks like.

Deepti Dabral , the purpose of Native PAGE to observe migration of proteins and their complexes or oligomers in their native state. Heating any protein would denature the protein and thus it can't form oligomers or bind to anything. Proteins are heated in SDS PAGE to denature the proteins so that once they bind SDS the proteins are negatively charged and their migration in the gel does not depend upon their folded state, but basically just their mass.