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I did Laemmli-SDS-PAGE for my Ammonium sulphate precipitate but I had very weak band and have very weird part at the end of gel. Please help me to solve that problem. Thanks enter image description here

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closed as unclear what you're asking by David, theforestecologist, AliceD Mar 7 '18 at 23:13

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    $\begingroup$ Did you remove the ammonium sulfate by dialysis or similar? $\endgroup$ – canadianer Mar 6 '18 at 15:57
  • $\begingroup$ No, I did not. I precipitated by AS and used PBS to dissolve, added buffer and loaded on gel $\endgroup$ – Huong Mar 7 '18 at 8:56
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Your gel looks just fine to me. It simply indicates that your ammonium sulfate precipitate contains massive amounts of this small protein at the bottom (whatever this is). The upper bands look fine, the amount loaded is appropriate: they are not overloaded like the bottom one, but not too faint either. The fact that your first and last lanes look a bit smeary is because of the massive amount of the small protein (lanes do look smeary when overloaded this much). The bottom band is definitely really some protein, and not a staining artifact; I have seen this shape before with purified proteins overloaded on SDS-PAGE.

One thing you could do, assuming you still have enough of these samples, is to run a complementary gel with 100 times less material. Dilute by a factor of 1/100 in 1X Laemmli denaturation buffer, and load a new gel with the same volume as for this one. You won't see the upper bands anymore, but you will have a much cleaner bottom band that you will hopefully be able to assign an apparent molecular weight to (or even cut out for mass spec identification; wear gloves and use clean tools if you do that, otherwise they will mostly detect keratin from your skin).

Assuming the point of this gel was to check the outcome of ammonium sulfate precipitation used as a purification step, well obviously it didn't work well to separate all these bands, and you might need to repeat and try different concentrations. But if the bottom band is the one you are trying to purify, then you were successful (the top bands represent less than 10% contamination, I would say by eyeball estimation). If you're interested in one of the top bands, then it clearly didn't work.

Is one of these lanes the supernatant? If so, you also have massive amounts of the small protein there (it's in every lane). Such a high enrichment looks suspicious: could that be purified lysozyme that you added during the purification procedure to help break down bacterial cell wall? (assuming you're trying to purify a protein over-expressed in E. coli).

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  • $\begingroup$ Thanks you for your respond. I would like to determine the molecular weight of the bottom band, because when I overlaid the half of gel with pathogens, the place of inhibition zone is the bottom of the gel (the second lane, beside ladder) . I will try to dilute the buffer as you suggest. Thanks $\endgroup$ – Huong Mar 7 '18 at 9:53
  • $\begingroup$ OK, so if you're interested in the bottom band you can definitely dilute (I suggested by 1/100, but you might need to try a few different dilution factors until you get a reasonable band). You can also use a denser gel, to make this band migrate slower (in this gel, it looks like this band is in the migration front). What was the percentage of this gel? Also, if the answer helped you, please mark it as accepted. :-) $\endgroup$ – Guillaume Mar 7 '18 at 15:50
  • $\begingroup$ Hi, I tried to dilute it to several different dilutions but it was not different as before, I did not get a reasonable band. The percentage of this gel is 4- 20%. I think maybe the size of this compound is smaller 11KDa (because I used Color Protein Standard marker, 11-245 KDa) or this compound is not protein, like lipid $\endgroup$ – Huong Mar 13 '18 at 8:36

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