Difference between revisions of "Antimicrobial Peptide Extraction"
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== Materials Needed == | == 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 == | == Protocol == | ||
| Line 20: | Line 66: | ||
-Discard the supernatant and let the protein precipitate dry overnight | -Discard the supernatant and let the protein precipitate dry overnight | ||
| − | -Wash the protein out of the centrifuge tubes with 35% Acetonitrile into a plastic tube | + | -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 [[High Performance Liquid Chromatography|HPLC]] | ||
===Mutacin 1140 extraction=== | ===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 [[High Performance Liquid Chromatography|HPLC]] | ||
== Notes == | == Notes == | ||
Latest revision as of 14:25, 20 July 2015
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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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