Antimicrobial Peptide Extraction
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Contents
Purpose
This protocol is used for extracting antimicrobial peptides made by bacterial species.
Materials Needed
Occidiofungin extraction
-Potato dextrose broth
-Burkholderia contaminans
-large centrifuge tubes
-centrifuge
-35% Acetonitrile
-2000 mL flasks
-stir bar
-stir plate
-Sodium Thiosulfate
-plastic pipette
-microfuge tubes
-microfuge
Mutacin 1140 extraction
-THYEX broth
-Streptococcus mutans
-large centrifuge tubes
-centrifuge
-35% Acetonitrile
-2000 mL flasks
-chloroform
-plastic pipette
-microfuge tubes
-microfuge
Protocol
Occidiofungin extraction
-Culture the Burkholderia contaminans bacteria in Potato Dextrose Broth at 25 C for at least 5 days
-Centrifuge the bacteria at 13,000 rpm for 20 minutes
-Pour the supernatant into 2000 mL glass flask with a stir bar and add 0.5 volume of Sodium Thiosulfate (i.e. 750 mL of supernatant, 375 grams Sodium Thiosulfate)
-Stir the mixture for 2 hours
-Centrifuge the mixture at 13,000 rpm for 20 minutes
-Discard the supernatant and let the protein precipitate dry overnight
-Wash the protein out of the centrifuge tubes with 35% Acetonitrile and a plastic pipet into a plastic tube
-Distribute the contents between microfuge tubes and centrifuge at 15,000 rpm for 10 minutes
-Extract the supernatant with a micropipette and place into a fresh tube for further purification in HPLC
Mutacin 1140 extraction
-Culture Streptococcus mutans to saturation in THYEX broth media
-Pour an equal volume of chloroform into the flask containing the cultured bacteria and let sit in fumehood overnight
-Pour out the upper broth layer and centrifuge the bottom chloroform layer at 13,000 rpm for 25 minutes
-Drain the chloroform, leaving behind the white semi-solid
-Centrifuge again at 13,000 rpm for 25 minutes
-Pour out the supernatant and let the precipitate dry overnight
-Wash out the precipitate with 35% Acetonitrile and a plastic pipet into a plastic tube
-Distribute the contents between microfuge tubes and centrifuge at 15,000 rpm for 10 minutes
-Extract the supernatant with a micropipette and place into a fresh tube for further purification in HPLC
Notes
Papers where this or a similar method has been used
Related Ontology Terms
References
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