Table of Contents

Phenol-water extraction (200-600 mL scale)

Introduction

Phenol-water extraction separates proteins from bacterial extracts. The principle for the separation is that a 50/50 mixture of phenol and water is heated to 65-68 deg C at which temperature only one homogenous phase is present. After cooling two phases are obtained and the polysaccharides and nucleic acids are in the water phase and the proteins in the phenol phase. A totally insoluble fraction mostly cell remains are obtained in either the inter-phase or in the bottom if centrifuged hard. Sometimes the polysaccharide goes to the phenol phase.

Reagents

Full list of chemicals

Procedure

  1. Suspend 20 g bacteria in 350 mL of hot water (65-68 deg C).
  2. Add 350 mL of 90% phenol (65-68 deg C) under vigorous stirring.
  3. Stir for 1h at 65-68 deg C.
  4. Cool in an ice bath to ca 10 deg C, or leave overnight in refrigerator.
  5. Separate in separatory funnel if possible, or centrifuge at 3000 rpm 30 for 45 min, or pour everything in dialysis bag.
  6. The upper, water layer is saved (not the interphase).
  7. Treat the residual phenol phase and interphase, if any, with another volume of hot water and proceed as described.
  8. Dialyse the combined water extracts 2-4 days in first tap water and then distilled water.
  9. Concentrate to a volume suitable for freeze-drying and freeze-dry.

Comments

  1. Sodium salts of LPS do not dissolve in H2O but form micelles and give a colloidal mixture.
  2. 90% aqueous phenol is a liquid and there much more convenient to handle than solid phenol. The separation of LPS from cell wall is mostly requiring stirring at high speed. If the stirring is too efficient foam will be formed and the total volume increase. In many cases loss of material will result.
  3. Sometimes only 15-30 minutes is required for the extraction.

References