I'd like to establish how resistant a certain protein is to denaturing by running native PAGE gels of the protein incubated at different pHs. As a proof of concept, I'd like to run a gel of catalase incubated at pHs 5, 7, and 9. I envision using sodium phosphate buffers with identical salt concentration and incubating for an hour, and then running one native PAGE gel comparing the three. Any tips for native PAGE are also appreciated. Thanks
The range of pH you intend to use is a bit too wide for sodium phosphate buffers. You may have to use different buffer salts to cover the whole range.
Personally, rather than running gels I would prefer to do a colorimetric assay on the treated samples using a spectrophotometer. It is more quantitative. The rate of catalysis tells you how much active enzyme is present. Of course, the rate of catalysis also is affected by pH, so after incubating the protein at different pHs, dilute all the samples into the same buffer for the assay.
Watch out for recovery of activity due to refolding of denatured protein when it is diluted into an optimal pH. This will show up as a gradually increasing rate of catalysis rather than a constant rate.
The range pH you intend to use at acid ( sodium acetate) between 4 and 5 ; and tris-HCl at pH (6-7) , and at Tris-base (8-9). after at you can running gel-electrophoresis.
Sodium phosphate buffer range is 5.6 to 8 so in this range you can choice pH 6 , 7 and 8
used the same salt concentration for the three then run native gel for the three see what you observed also you can measure activity of equal amount of catalase at those three pHs in this way you see the effect of pH on activity and mobility in native PAGE good luck
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