I need to run my protein in a Native PAGE. It's pI is 8.16. I studied Native PAGE protocols and they are very vague about the upper limit that a protein pI must have in order for it to run to the cathode. Some protocols say 8, some says, 8-9. Since my protein is in the limit I wanted to know if anyone here had the experience of running a Native PAGE with a protein of similar pI. Should I run it to the cathode (like SDS-PAGE) or the anode (inverting the poles of the power supply)?
Similar problem was faced in our lab and we solved it by reversing the electrode. If size of your protein is above 50 kDa run a 10% Native PAGE.
Part of the approach depends on the sample: is this a pure protein? a crude extract? My general approach is to have the protein migrate as far as possible, which should (with caveats) give you the best chance at high resolution of separation from closely similar molecules. If you don't migrate far enough, you may not get much spatial discrimination -- thus not much resolution. Also keep in mind that the loading buffer pH may influence the outcome, since you can alter the starting charge on the protein.
All that said, I would just try the pragmatic approach: run one gel each 'direction', and see which outcome you're happy with. Ultimately, you'll never know if you guessed right anyway unless you have comparison for better or worse. There are enough unknowns in combination with sometimes unpredictable operational variables that some quick tests/comparisons are worthwhile.
very simple. You just try to use both methods, run to the cathode and anode, and it will prove which is right.
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