Consider using clear-native PAGE. It performs much better when analysing basic proteins than BN-PAGE. In the attachment you will find a paper that gives you more information.

Cheers, Christian 

Article Advantages and limitations of clear-native PAGE

Christian Q. Scheckhuber, thank you very much for your reply.

I am familiar with this paper, but have not noticed before that here they are running CN-PAGE by simply changing the cathode and loading buffer. 

However, I do not quite see how this would help with my problem - without the Coomassie in loading dye and cathode buffer to impart negative charges it might even make it worse? I would imagine that under normal running conditions my protein wouldn't even stay in the wells in this CN-PAGE setup.

Hi! I had a similar problem while running BN-PAGE for recombinant membrane proteins. I got better results by changing the sample buffer with Ponceau red (5% glycerol and 0.01% Ponceau-Red). I hope that this might work for you as well. 

Article Binding Specificity of Retinal Analogs to Photoactivated Vis...

Sundaramoorthy Srinivasan, thank you for your helpful reply. I will certainly give this sample buffer a shot, but I notice that the pI of rhodopsin is around ~6. Did you have the same problem with your protein "sticking" to the wells?

@Nadia: Of course you are right. CN-PAGE is restricted for use with acidic proteins. I was wrong when I said that it works better with basic proteins than BN-PAGE. Sorry!

Regards, Christian 

Christian Q. Scheckhuber No problem Christian, it's only good for me to get an oppurtunity to check my thinking.

Do you think that increasing the voltage could help "push" the proteins into the gel? I use the Invitrogen/Novex system which simply states to run the Bis-Tris gels at 150V.

Do you think that adding more of the Coomassie sample loading buffer could help? I am assuming that my proteins at some point are "saturated" by negative charges, but could it be that is "falls off" the loaded protein during the run (~2 hours)?

Increasing voltage or Coomassie concentration won't help you in my opinion. By doing so you could affect the quaternary structure of your protein of interest. Maybe you could try CN PAGE and simply switch the electrode leads at the power supply. Then basic proteins like connexin should be able to enter the gel because they are now migrating to the cathode. You would lose the acidic (and probably neutral) proteins, though.

I hope this information might help you!

Regards, Christian

Yes! at least the mobility was very slow.. The gel system and running buffer that I used, were different than yours.

Christian Q. Scheckhuber I did give "switching" the electrodes on the BioRad Criterion system (TGX gels) a shot, but it seems I lost all the protein (although I did see the loading dye go "upwards" and out of the gel as I should). I ran it at 200V for 55min (as per the Criterion manual) and used a loading dye without Coomassie (basically glycerol and some BBB to see the sample).

I will try reversing the electrodes once more on the Invitrogen/Novex system and post any updates here.

I will also try a BN-PAGE with the tips you gave me, Sundaramoorthy Srinivasan, although I'm thinking that it won't help my protein due to the high pI (~9).

I promised to update you if I had any progress. Sadly (but not surprisingly), changing the sample buffer did not help.

1) Clear Native PAGE with reversed electrodes: my protein did not enter the gel, it was stuck in the wells.

2): BN-PAGE: I decided to prepare new PM samples and I ran my experiment under the same conditions (exactly as Invitrogen recommends) with one change: I stored my samples in the fridge (4C).

In this case, my samples successfully ran and I did not observe aggregates in the wells. The gap junction band still shows smearing, but this might be due to several reasons, including possible heteromeric gap junctions. However, the bands were not "constricted" as you see in the image above, but rather ran normally through the gel.

I suspect that at least in this case, freezing of the sample caused aggregation.

The aquaporin did not seem to be negatively affected by freezing, which is probably why it took me so long to check for freeze-thaw effects.

I hope this update can be useful for others working with basic proteins in BN-PAGE.

Hi Nadia,

you may try out the addition of "very low" concentrations of SDS (or LDS) to the cathode buffer and/or to the sample/separation gels; e.g. 0.001% up to 0.005% (conferring negative charge).

In quite a lot of cases, protein complexes are sufficiently stable to survive these conditions (or even little harsher ones). You may try this out both for CN and BN-PAGE as platform techniques. I remember that there are corresponding references available, as such a protocol may also result in sharper bands for "well-behaved" proteins.

Simultaneous addition of DDM/digitonin (as the optimal detergents for solubilisation of your target protein complexes) to cathode buffer and/or gels may be helpful as well. In this respect, addition of a low DDM concentration to CN gels corresponds to the original CN-PAGE recipe published by Schägger (then named as Colourless-Native PAGE). Similarly, addition of mild non-ionic detergents to the CN-PAGE cathode buffer represents high-resolution CN-PAGE (http://www.mcponline.org/content/6/7/1215.long). Since electrophoresis is to a large extent an empirical endeavour, you should feel free to modify the systems thoughtfully. For this, you may make use of the basic knowledge generated by pioneers as Andreas Chrambach and others.

Best regards,

Frank

Hello Frank, Thank you for your informative answer.

I did actually attempt your suggestions with addition of low concentrations of SDS. Unfortunately, it didn't seem that my protein of interest migrated in BN-PAGE under any of the conditions available to me.

I assume this is due to the high pI (~9) and the tendency of connexins to aggregate (which I have previously observed in protein lysates from transfection experiments).

We have since moved on and used a different method for this experiment. However, I will keep your suggestions in mind for further work.

Best regards,

Nadia

Hi all,

I also encountered problems with blue native gel. My samples were E. coli-purified rProtein in buffer containing

10mM Tris-HCl pH8.0, 150mM NaCl (pI± 7.4, 27kDa). I ran the commercial available 4-16% Bis-Tris gel (Thermo) with commercial buffer (Thermo). First trail was nothing (or a little somethings) in the gel. Then I added some Coomassie G-250 in samples and ran only with dark blue buffer. There were something smearing but not sharp bands. (I loaded a lot of protein in a lane) Please see the conditions and results in the attachment. Could anyone help me as well? I am desperate. :(