Nurun and Hiasindh,

Your sample buffer will certainly appear to be running out of the gel when you invert the electrodes. This is exactly what is supposed to happen. In a typical native gel, the bromophenol blue (which is acidic) along with the acid proteins run into the gel. Your basic protein runs out of the gel, which is why you can't see basic proteins on a regular native PAGE gel. When you invert the electrodes, you are trying to get the basic proteins to run into the gel, and the acidic components (including the bromophenol blue) will be running out of the gel. This is excpected and should not cause any alarm. In fact, this is an indicator that the leads have been reversed appropriately.

Your basic proteins are neutrally charged at high pI (9.6 and 10.4, in your cases). They are positively charged at pHs that are lower than the pI. In a standard SDS-PAGE, the the cathode (negative) is at the top of the gel and the anode (positive) is that the bottom of the gel. The pH of the gel is lower than your proteins' pIs, so your positively charged basic proteins run out of the gel under these conditions. You can force your basic proteins to run into the the gel under two conditions. One condition is if you use the standard electrode set-up (negative cathode at top, positive anode at bottom) and you are able to pour a gel has a pH that is HIGHER than the pI of your protein. Your basic protein would eventually become negatively charged and run towards the traditional bottom of the gel if you could get the pH of the gel high enough. Your proteins are bot very basic so this is not a good solution. The second option is to invert the electrodes and run the gel at a lower pH. At low pHs, you basic protein will be very positively charged. BUT, in order to get the positively charged protein into the gel, you have to put the positive electrode at the top and the negative electrode at the bottom (i.e., you have to switch the electrode leads at the power supply).

Also, Hiasindh, there are other options besides b-alanine. See http://www.aesociety.org/areas/pdfs/Garfin_1DE_WebArticle9-07.pdf for a more complete list of possibilities for the McLellan buffers. Starting on page 10, you will see the acidic and basic components. You could use gaba or histidine at the appropriate concentrations (also see the necessary acidic components). Also, look at Figure 4 and check out the note in the Figure 4 legend about reversing the electrode leads for gels under pH 7.4. The lowest pH gel will be the gel in which your basic protein will migrate fastest. I suspect that with a protein pI ~ 10.5, even a gel of pH 5 of 6 would will still allow your protein to migrate into the gel. Instructions for casting a range of gels with McLellan buffers follow on pages 11-12 and there is a short paragraph on basic proteins on page 13. Hope this information is helpful.

try using Nupage Native gels and follow their protocol. it works

Refer to Protein volumes 1-5 for electrophoresis it may give an idea because the protein MW is 3400, so very small protein. Is it associated with any other protein? Do you have probes to identify for raw (crude) protein? Perform western blot after electrophoresis, if not run the gradient gel from 15% to 25% PAGE.

Thanks for your answers. Its not associated with any protein or crude extract. Scine its transmembrane domain of a protein, i have synthezied that region alone with 95 % purity. I want to study the oligomeric state of the peptide using native page. Any help regarding this?

if it's transmembrane, than Blue-native or clear-native PAGE should work best probably.

I ran 15 % Tricine Native PAGE and did silver staining. But, it din't run into the separating gel. I found band inbetween the stacking and separating gel.

You might try running a typical native PAGE but when you put the electrodes into the power supply, invert them (i.e., put the red electrode into the socket for the black power supply and black electrode into the socket for the red power supply). Basic proteins will not migrate into a standard PAGE gel because only proteins with lower pIs (say < 7-8) will migrate under native conditions. Acidic proteins are negatively charged and will migrate into a standard native page, but basic proteins are positively charged and will not migrate into a standard native page...basic proteins migrate OUT of a standard native page. If you flip the electrodes, you reverse the electric field, and this will allow basic (i.e., positive) proteins to run into the gels. BioRad recommends this approach for native-PAGE of basic proteins (see their manual for TGX mini-gels under "native-PAGE". This should work with any standard native gel type, whether it's BioRad's gels, some else's gels, or hand-case gels.

Hi, did you get a solution for your problem? I am also facing the same problem, my protein's pI is 9.6. Did you try inverting the electrodes? I was trying but when I inverted the electrodes I saw the sample buffer is running out, I got panicked and change the electrode in the right way.

Hi Nurun,

It happened for me also, when inverting the electrode, the sample buffer came out. So, i changed the electrode as we run usually. But didn't work out. I am trying with different procedures. If anything comes properly, will let you know.

Nurun and Hiasindh,

Your sample buffer will certainly appear to be running out of the gel when you invert the electrodes. This is exactly what is supposed to happen. In a typical native gel, the bromophenol blue (which is acidic) along with the acid proteins run into the gel. Your basic protein runs out of the gel, which is why you can't see basic proteins on a regular native PAGE gel. When you invert the electrodes, you are trying to get the basic proteins to run into the gel, and the acidic components (including the bromophenol blue) will be running out of the gel. This is excpected and should not cause any alarm. In fact, this is an indicator that the leads have been reversed appropriately.

Your basic proteins are neutrally charged at high pI (9.6 and 10.4, in your cases). They are positively charged at pHs that are lower than the pI. In a standard SDS-PAGE, the the cathode (negative) is at the top of the gel and the anode (positive) is that the bottom of the gel. The pH of the gel is lower than your proteins' pIs, so your positively charged basic proteins run out of the gel under these conditions. You can force your basic proteins to run into the the gel under two conditions. One condition is if you use the standard electrode set-up (negative cathode at top, positive anode at bottom) and you are able to pour a gel has a pH that is HIGHER than the pI of your protein. Your basic protein would eventually become negatively charged and run towards the traditional bottom of the gel if you could get the pH of the gel high enough. Your proteins are bot very basic so this is not a good solution. The second option is to invert the electrodes and run the gel at a lower pH. At low pHs, you basic protein will be very positively charged. BUT, in order to get the positively charged protein into the gel, you have to put the positive electrode at the top and the negative electrode at the bottom (i.e., you have to switch the electrode leads at the power supply).

Also, Hiasindh, there are other options besides b-alanine. See http://www.aesociety.org/areas/pdfs/Garfin_1DE_WebArticle9-07.pdf for a more complete list of possibilities for the McLellan buffers. Starting on page 10, you will see the acidic and basic components. You could use gaba or histidine at the appropriate concentrations (also see the necessary acidic components). Also, look at Figure 4 and check out the note in the Figure 4 legend about reversing the electrode leads for gels under pH 7.4. The lowest pH gel will be the gel in which your basic protein will migrate fastest. I suspect that with a protein pI ~ 10.5, even a gel of pH 5 of 6 would will still allow your protein to migrate into the gel. Instructions for casting a range of gels with McLellan buffers follow on pages 11-12 and there is a short paragraph on basic proteins on page 13. Hope this information is helpful.

Hi, All I am also facing the same problem. I have a protein complex one with positive charge and one with negative charge. But I want to run it on native gel to check shift for confirming the Interactions. I also tried reversing the electrodes, Running Acidic native gel but anything did not worked. Any Suggestions?