I am a member of undergraduate research anticipating to investigate the protein expression of fungal biofilm after teatment of shockwaves and amphotericin B. After growing biofilm and containing its extracellular matrix in Tris-HCl buffer, a series of treatments, vortexing, ultrasonication, treatment with cation/anion exchange resins, centrifuging, and filtering with millipore membrane will result in supernatant that contains proteins, triglycerides, and carbohydrates. Unfortunately, we are unsure how to isolate proteins from the supernatant containing other organic compounds. Any feedback on how we can successfully isolate pure ECM proteins from the supernatant will be greatly appreciated.
In addition, if there are other successful protocols for extracting and isolating proteins from fungal biofilm ECM, it will also be very much appreciated.
As you are using Tris-HCl, I assume your buffer pH is >7. Many, actually proteins bind anion exchangers at pH values of pH 8 - 9. A smaller fraction will bind cation exchangers at pH 5 - 6.
But as proteins are large, you might need to use different ion exchangers than the ones you are probably using for small molecules; their pore sizes are too small for proteins.
A good start and quite cheap, as it is no longer patent protected, is Q Sepharose Fast Flow (Anion exchanger); the cation exchange version is SP Sepharose FF. . You can find it in the catalogs of many suppliers.
Hi Nanasi,
The link below might be useful -
https://www.researchgate.net/post/Any_good_protocol_for_isolation_of_extracellular_matrix_ECM_proteins_from_liver
All the best!
Triglycerides are insoluble in water so those can be separated with liquid-liquid extraction using an organic phase and HPLC isn't necessary for this. See the attached article. I don't think you should used EDTA like they did, though.
Removing carbohydrates seems difficult because carbohydrates are soluble in water. Searching density of carbohydrates only gives energy density which is completely useless. But the density of some common biological carbohydrates like glucose, glycogen, starch is generally greater than that of protein (1.35 g/mL). So I think you could separate the carbohydrates with density gradient centrifugal. But please use a salt, don't use sucrose like common practice. That would screw everything up probably.
H.G. Zha, Bubacarr G Kaira , Adron Ung , Achim Recktenwald , thank you all so much your insight and feedback to our question is very valuable and definitely adds more factors for us to consider for the course of our experiment. Again, many thanks!
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