The Gel has pH 8.9. and the pH of loading dye is 6.67. What should be the pH of running buffer to allow smooth running of my protein into the gel.
I prepared buffer of pH 8.49 but I was unable to see my protein in the gel.
If the pH is below the pI, the native protein will be net positively charged and will move toward the anode (negative or ground electrode). If the pH is above the pI then the protein will be net negatively charged and move toward the cathode (positive electrode). You have picked the worst possible system with a loading buffer below the pI and a running buffer above the pI. Stick with a single pH above or below depending on the electrode configuration (i.e, adjust your loading buffer to pH 8.9.
You can use native PAGE on acidic conditions .
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In both SDS-PAGE and cationic PAGE, the pI of the protein is irrelevant. The charge of the bound detergent molecules (about 1 per 3 amino acids) is by far greater than that of the charged groups of the protein itself. That is the reason for adding the detergent: all proteins get the same charge/mass ratio and hence the same acceleration in an electric field. However, the interaction of proteins with the gel network is size-dependent.
In native (detergent-free) electrophoresis, of course, your pH should be different from the pI of the protein (about 1 unit, at least). There are standard systems available for DISK-electrophoresis at different pH, check for example https://patents.google.com/patent/US3620947A/en or doi:10.1002/elps.1150040303.
Thank you so much, everybody. I have switched to Blue-Native PAGE. The recipe of Gel formation is by doi:10.1038/nprot.2006.62. However, the stacking gel (3.5 %), does not solidifies, even after two hours. Could anybody share an experience with BN-PAGE.
A 3.5% acrylamide gel is almost liquid. If you really need that low a concentration, add 0.5% agarose (electrophoresis grade). This will solidify the gel, but agarose pores are so wide that whole virus pass through. Note, however, that these pores form only during curing of the gel in the fridge at least o/n.
Agree with Buxbaum's answer. 3.5% acrylamide is tough to work with. 4% is OK. Be sure that you have degassed the solutions thoroughly before use because O2 interferes with the polymerization. The best way to degass is to vacuum filter the monomer solution through a 0.2 or 0.45 micron filter. Before adding the TEMED and persulfate. Use it immediately and mix gently so as not to introduce air and bubbles.
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