3
$\begingroup$

enter image description here

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.

$\endgroup$
  • $\begingroup$ @vajra78 Each sample is a pure protein. $\endgroup$ – canadianer Mar 22 '15 at 7:22
  • $\begingroup$ what exactly do you mean with 'resolve'? "To get a clear band"? Why is B better (i.e., clear band) than A you think? Isn't A not just totally degraded? $\endgroup$ – AliceD Mar 22 '15 at 12:04
  • $\begingroup$ @AliceD Yes, get a clear band. I have no idea why B is better than A, the only difference is the FLAG tag (ie greater charge:mass ratio). A is not totally degraded because I can resolve it with SDS-PAGE. $\endgroup$ – canadianer Mar 22 '15 at 17:55
  • 3
    $\begingroup$ When I see a smear I suspect heterogeniety. Since the smear goes up, it suggests the proteins are heavier (though it's a native gel and you can't assume mass and migration are directly correlated). Is there some possibility that A is polymerized? And the FLAG tag on B somehow disrupts this polymer? $\endgroup$ – user137 Mar 22 '15 at 18:38
  • $\begingroup$ @user137 That's an interesting idea; I was thinking it was just a problem with my gel conditions. The protein should have no specific interactions with one another. However, each monomer has a free cysteine that could possibly form a disulfide bond with another monomer and link the tetramers together. The FLAG tag could just add some electrostatic repulsion to prevent close contact of the cysteines. It's kind of a nice explanation; however, I really don't expect it to happen readily. I'll add some mercaptoethanol to the loading buffer and see what happens. $\endgroup$ – canadianer Mar 22 '15 at 18:53

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.