A practical guide to structural analysis of carbohydrates

Removal of protein using proteinase K

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


  1. 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.
  2. Add 1 mG of Proteinase K and leave at room temperature for 16 h.
  3. Optional: Ultracentrifuge at 100 000 g for 4 h.
  4. 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.