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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.

Kushal
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  • There are many methods to concentrate protein depending on the protein itself, volume, resources availble, etc. Perhaps you could provide some more details. – canadianer Mar 31 '18 at 21:15
  • 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. – Kushal Apr 01 '18 at 17:08
  • 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. – Kushal Apr 01 '18 at 17:09
  • It sounds like you already have a concentration step. Can you resuspend the pellet in less urea? – canadianer Apr 01 '18 at 17:11
  • I already did in the most minimal amount possible. I dont think less than that would be sufficient to dissolve the pellet. – Kushal Apr 01 '18 at 17:53
  • I have thought of using glass beads and ammonium carbonate buffer together. Instead of filteration what if we centrifuge the broth collect and wash the pellet. suspend pellet with water and glass beads with vortexing it for 15 min. then collect the suspension and add 1ml of it for 10ml buffer for overnight followed by methanol precipitation.....do you think this can work? as I am not sure about it....and if it does, when we add distilled water along with glass beads will the protein get diluted instead? – Kushal Apr 01 '18 at 18:00
  • another alternative which I think is collecting the protein supernatant and preciptate it again by methanol..centrifuge it...but I dont know if this step gives any pellet.. so if pellet is obtained dissolve it again in urea+DTT..centrifuge to get the protein supernatant...but my query in this protocol is that will the earlier urea+DTT used to dissolve the pellet earlier interfare with methanol precipitation and affect my proteins? – Kushal Apr 01 '18 at 18:06

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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.
tyersome
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  • 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! – tyersome May 28 '21 at 17:29
  • 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. – tyersome May 28 '21 at 17:47