RNA is a common contaminant in LPS preparations due to cell rupture and leakage of RNA.
Ribose appearing in a hydrolysate (sugar analysis) is most
often a sign of contaminating RNA. RNA may be removed by treatment with RNAse or alkali,
both followed by purification by gel filtration or dialysis.
- Ribonuclease A
- 0.1 M CaCl2
or MgCl2
- 0.1 M Sodium acetate buffer
Procedure
- Dissolve the LPS in 0.1 M acetate buffer (pH 5) to 20 mg/mL (an opalescent
solution may be obtained) or take solution direct from dialysis.
- Add 1 mL of 0.1 M CaCl2
solution per 100 mL.
- Add 0.4 mg RNAse per mL LPS solution.
- Leave at 37 °C for 3-4 h or in a dialysis bag at 22 °C over
night, in either case with 1 drop of CHCl3
.
- Desalt by gel filtration or dialysis over night.
- Freeze-dry.
Comments
- Chloroform is added as an antibacterial agent.
- If phenol extraction has preceded, it
is possible to concentrate the solution and run directly.
- The enzyme is normally such a
small amount that it can be neglected. If desired it may be removed by conventional methods,
e.g. phenol extraction.
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