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I need to isolate a protein that a bacteria has excreted into an agar plate. My plan is cut out parts of the agar, heat the agar to melt it and then concentrate the protein through some purification kit, and then run SDS-PAGE to see if I was successful in isolating the protein.

However, I am not sure whether heating the unknown protein before SDS-PAGE will effect the gel run in anyway. Does anyone know? I know if might denature the protein in some way, but I am doing that anyways with SDS.

Thanks.

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  • $\begingroup$ I think this depends strongly on the kit you are using for the purification. Agarose doesn't need to be heated until boiling, usually they get liquid around 60°C. Or you could try to use some low melting agarose in a higher concentration. These melt around 37°C. $\endgroup$ – Chris Nov 21 '16 at 21:10
  • $\begingroup$ I guess the next question is what is the most effective way of extracting proteins from LB agarose. Are there any pre-defined methods for this? $\endgroup$ – Adam Radek Martinez Nov 22 '16 at 14:41
  • $\begingroup$ Not that I know of. It also depends on your agarose. $\endgroup$ – Chris Nov 22 '16 at 15:09
  • $\begingroup$ I will have to be using LB Miller Agar $\endgroup$ – Adam Radek Martinez Nov 22 '16 at 15:12
  • $\begingroup$ Could you just make a liquid subculture and isolate your protein from the medium? $\endgroup$ – canadianer Feb 3 '17 at 22:08
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As far as I know, it has an effect. If you denature your protein before running the gel, you do a normal SDS-PAGE and seperate the proteins by their size. Since you use SDS, the charge of the protein doesn't have any influence on your gel.

If you don't heat your proteins before, you do a so called native PAGE. Here the size of your protein doesn't matter so much, because you also seperate for charge, folding, pH and so on. You may have a look on this methods paper for more information.

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  • $\begingroup$ It's native PAGE, using SDS is by definition not native. I guess a typing error? $\endgroup$ – VonBeche Nov 21 '16 at 21:02
  • $\begingroup$ I changed it to native PAGE :) But SDS is the most common used, so the most will understand it. $\endgroup$ – SeRe Nov 21 '16 at 21:04
  • $\begingroup$ It's not the heating per se that separates native from SDS PAGE. It's the SDS in the gel and the running buffer and the presence of denaturing agents in the preparation of the samples. Native gels you do after you know a few things about your protein and you want to get more information. But the first step in isolating and potentially identifying a protein from a culture (among SDS and native gel) is most probably the SDS PAGE as you would be able to separate all the proteins by size and as monomers. $\endgroup$ – BioGeo Nov 21 '16 at 21:16
  • $\begingroup$ If I use regular PAGE, how do I extract the protein from the agarose that the bacteria are growing on? $\endgroup$ – Adam Radek Martinez Nov 22 '16 at 14:42
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It would depend on the temperature and how long you keep it there. For SDS-PAGE we normally do it at 95 degrees Celcius for 5-10 minutes. Keeping it much longer could degrade the protein and fragment it.

You could try to do your culture on agar of lower concentration. Alternatively, you could aim for liquid cultures, as it would facilitate handling before the SDS-PAGE.

(The whole story changes if you expect your protein to be heat-tolerant - as it could be the case that your bacterium is a thermophile)

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  • $\begingroup$ I am not sure. I am seeing a zone of clearance around the colonies in the presence of silver, which makes we suspect that some exoenzyme is being released. $\endgroup$ – Adam Radek Martinez Nov 22 '16 at 15:14
  • $\begingroup$ You mean you add silver in the agar? Can you add it in a liquid culture and see what extracellular enzymes you can detect in the SDS PAGE afterwards? $\endgroup$ – BioGeo Nov 23 '16 at 20:23

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