This is a native gel. Let's call the left 2 lanes protein A and the right 2 lanes protein B. B is the same as A except it has a FLAG tag. They are both homotetramers of about 65 kDa. After purification, both appeared as single bands on SDS-PAGE. However, these are relatively old samples and I expect multiple bands due to degradation (which I've observed with previous samples). Either way, I cannot for the life of me resolve them with native PAGE. B is somewhat workable, though still atrocious. For each of A and B, I've loaded 5 and 15 uL (hence why there are two lanes for each) with 5 uL of 5X loading buffer. Yes, I know that's not the correct ratio; I can't imagine that being my problem but I'm running it again with the correct ratio. The rest of the conditions are pretty standard:
- The protein was in PBS (50 mM dibasic sodium phosphate, 75 mM NaCl, pH~7.3) prior to adding loading buffer; I've also used TBS (50 mM Tris, 7.5 mM NaCl, pH~7.4) with similar results
- 4% stacking gel, pH=6.8
- 6% resolving, pH=8.8
- Tris-glycine running buffer, pH~8.3, ran at 4°C with 23 mA constant current. I've also ran at 30 mA with similar results.
- Stained with Coomassie
Let me know if any other info is needed. Any advice will be greatly appreciated.