Proteins are common contaminants in saccharide preparations. The quantity of protein
can easily be estimated with the Lowry method or a modification
such as the Bio-Rad protein assay. The removal of proteins may be effected in different
ways, phenol extraction being one and treatment with protein
digesting enzymes another. One of the best enzymes (albeit expensive) at present
is proteinase K. After the digest the enzyme can be removed or simply kept if it does not
disturb the next purification step. If purification is desired phenol extraction or gel
filtration may be used. As in most enzyme digests the temperature, pH and the buffer are
of importance. If the digest is proceeding for a long time sterile conditions are also essential.
- Proteinase K, 10-20 units/mg (Sigma)
- Phosphate buffered saline (PBS), 0.1M pH7.2, sterile
- Chloroform
Procedure
- Dissolve the sample, ca 50 mg of crude LPS preparation, in 2-5 mL PBS
and sonicate for 20-30 min in a sonicating bath.
- Add 1 mG of Proteinase K and
leave at room temperature for 16 h.
- Optional: Ultracentrifuge at 100 000 g for 4 h.
- If remaining Proteinase K is not a problem
purify by dialysis. To obtain a Preoteinase free preparation purify by gel chromatography
on e.g. Sephacryl.
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