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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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