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 °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, is obtained either at the interface or at the
bottom
if centrifuged hard. Sometimes the polysaccharide, in particular rough LPS, goes to the phenol phase which should be analysed for carbohydrates before being discarded.
- Stirrer ca 300 rpm, propeller
- 1L Duran bottle with wide neck.
- 90% aqueous phenol
Procedure
- Suspend 20 g bacteria in 350 mL of hot water (65-68 °C).
- Add 350 mL of 90% phenol (65-68 °C) under vigorous stirring.
- Stir for 1h at 65-68 °C.
- Cool in an ice bath to ca 10 °C, or leave overnight in refrigerator.
- Separate in separatory funnel if possible, or centrifuge at 3000 rpm 30 for 45 min,
or pour everything in dialysis bag.
- The upper, water layer is saved (not the interphase).
- Treat the residual phenol phase and interphase, if any, with another volume of hot
water and proceed as described.
- Dialyse the combined water extracts 2-4 days in first tap water and then distilled water.
- Concentrate to a volume suitable for freeze-drying and freeze-dry.
Comments
- Sodium salts of LPS do not dissolve in H2O
but form micelles
and give a colloidal mixture.
- 90% aqueous phenol is a liquid and there much more convenient to handle than solid phenol.
The separation of LPS from cell wall often requires 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.
- Sometimes only 15-30 minutes is required for the extraction.
References
- Meth. Carbohydr. Chem. V (1965) 83-91
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