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I am working with cell wall protein of Candida albicans and cryptococcus neoformans but after protein extraction and run on SDS PAGE my bands are not visible after coomassie staining. Can anyone tell me how to concentrate the protein so that bands can be seen on coomassie stain. I am using Ammonium Carbonate buffer for extraction of protein. Also, the bands are visible on silver staining.

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  • $\begingroup$ There are many methods to concentrate protein depending on the protein itself, volume, resources availble, etc. Perhaps you could provide some more details. $\endgroup$
    – canadianer
    Mar 31 '18 at 21:15
  • $\begingroup$ I am not working on particular specific protein. I just have to extract cell wall proteins of both and run on SDS PAGE and compare the bands using a marker. I am using Sabouraud Dextrose Broth as growth media for both of them. $\endgroup$
    – Kushal
    Apr 1 '18 at 17:08
  • $\begingroup$ I have inoculated each in SDB and kept for 7 days on shaker incubator and followed by filteration. This collected mycelia is stored at -20C. then I suspended 1g of mycelia into 10mL Ammonium Carbonate and left overnight on rocker shaker. Filtered it and precipitated with methanol (1:8) followed by centrifugation and collecting pellet. Then dissolved this pellet in Urea+ DTT(minimal amount) and microfuged to get protein in supernatant. But the protein is not visible on Coomassie staining after SDS PAGE. If you could provide any protocol it would be helpful aswell. $\endgroup$
    – Kushal
    Apr 1 '18 at 17:09
  • $\begingroup$ It sounds like you already have a concentration step. Can you resuspend the pellet in less urea? $\endgroup$
    – canadianer
    Apr 1 '18 at 17:11
  • $\begingroup$ I already did in the most minimal amount possible. I dont think less than that would be sufficient to dissolve the pellet. $\endgroup$
    – Kushal
    Apr 1 '18 at 17:53
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The problem here is concentration. Coomassie Blue stain can only detect protein band greater than or near to 50ng, in this case, the concentration of your protein is too low for detection. If you want to stick with Coomassie stain, you can try colloidal Coomasie stain instead because it has a much lower detection limit than Coomasie blue (between 10ng to 20ng).

In addition, I think what causes such low protein concentration is the poor solubility of yeast cell wall. One way to get around this is to increase solubility is through chemical hydrolysis, for example using diluted sulphuric acid1-3. This will partially solubilize mannas-protein outer layer of the cell wall and help you to obtain soluble yeast cell wall products.

References:
  1. Theodoro, S. D. S., Putarov, T. C., Tiemi, C., Volpe, L. M., de Oliveira, C. A. F., Glória, M. B. D. A., & Carciofi, A. C. (2019). Effects of the solubility of yeast cell wall preparations on their potential prebiotic properties in dogs. Plos one, 14(11), e0225659.
  2. OGAWA, K., & MATSUDA, K. (1994). Characterization of Manno-oligosaccharides Containing α (1→ 2)-and α-(1→ 6)-Linkages Obtained from Saccharomyces cerevisiae Mannan by Controlled Acid Hydrolysist. Journal of Applied Glycoscience, 41(4), 433-437.
  3. Peat, S. (1961). Polysaccharides of baker's yeast. Part IV. Mannnan. J. chem. Soc., 61, 29-34.
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  • $\begingroup$ Welcome to Biology.SE! This looks like a good answer and you've done a nice job of providing supporting links. However, in the future please include the complete reference information since links can break. One easy way to get that information is to search for the paper on Google Scholar and click on the ‟ symbol to get reference information. This is a good example of how to format references. I'm also not clear on whether your "this paper" reference is to that paper or to ref 25 of that paper. Thanks! $\endgroup$
    – tyersome
    May 28 at 17:29
  • $\begingroup$ I've converted your reference links into citations, but please check to make sure I have not make any mistakes and edit to correct/improve as needed. $\endgroup$
    – tyersome
    May 28 at 17:47

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