Hello everyone,
Recently I was running trastuzumab on native (tris glycine) gel from bio-rad in tris glycine running buffer (No SDS) pH 8.3. However, I didn't get any band on the gel. I checked the pl of trastuzumab and elevate the pH to 9.0 hoping that this will give enough negative charge but still it didn't work. The loading buffer was native loading buffer with BP. Could any one here give a helping hand?
Thanks in advance!
Reverse the polarity of the power running through the gel, that way your protein will enter the gel and be separated. This is what Biorad recommend for proteins with a pl above 8.5. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf#page=19
The resolution with Tris-glycine native PAGE will never be especially good as it is separating based on both size and charge. For better results, but a bit more work, look at using Blue Native PAGE (BN-PAGE) if it is available to you. This uses Coomassie G-250 in the samples to give a negative charge. You can buy the gels pre-cast from Thermo (they are called NativePAGE Bis-Tris Gels - https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/nativepage-bis-tris-gels.html). The buffers are a bit of a pain to make as the reagents take forever to go into solution, so it is way easier to just buy them, but they are fairly pricey (and even more since I last used them a few years ago). The recipes for the buffers are in the instructions for the gels though.
Maixian Liu great that it worked! I happened to remember that obscure paragraph in the Biorad TGX manual about reversing the polarity.
As you've probably found out, rudimentary clear native PAGE with tris-glycine gels isn't especially great. As I mentioned before, blue native PAGE is quite a bit better, but has its own problems. You should look up papers about so-called high resolution clear native page, which is a further improvement on blue native PAGE - see https://www.mcponline.org/article/S1535-9476(20)31690-X/fulltext for example. These use mixed micelles of a low concentration of sodium deoxycholate plus mild detergents like DDM, triton-X-100 or digitonin in the sample buffer and at a lower concentration in the running buffer. This will likely solve the solubility issue that you mentioned.
Unfortunately, these are not really compatible methods with conventional tris-glycine gels, you need to use gels that are based on imidazole or Bis-Tris. If you don't want to hand cast these, the same idea can likely be adapted to work with the pre-cast Thermo Native PAGE Bis-Tris gels I said about before (conventional Nupage etc bis-tris gels cannot be used for native PAGE, because they contain SDS in the gel), by replacing the commassie G-250 in the sample, anode and cathode buffers with the relevant mixed micelles described in the high resolution clear native PAGE. Although most high resolution clear native PAGE protocols typically use imidazole gels, I can't see any reason why the Thermo Native bis-tris chemistry gels can't be adapted to use the same idea of mixed micelles.
Good luck, if you decide to have a go with high resolution clear native PAGE, I'll be interested to hear/see how it turns out!
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