Antimicrobial Peptide Extraction

You can help OMP wiki by editing the content of this page. For information about becoming a registered user and obtaining editing privileges, see Help:Accounts.

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 use

References

See Help:References for how to manage references in OMPwiki.