RNAse treatment

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

Full list of chemicals

1. 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.
2. Add 1 mL of 0.1 M CaCl₂ solution per 100 mL.
3. Add 0.4 mg RNAse per mL LPS solution.
4. 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.
5. Desalt by gel filtration or dialysis over night.
6. Freeze-dry.

1. Chloroform is added as an antibacterial agent.
2. If phenol extraction has preceded, it is possible to concentrate the solution and run directly.
3. 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.