SIMPLE, SENSITIVE, AND QUICK PROTOCOL TO DETECT LESS THAN 1 NG OF BACTERIAL LIPOPOLYSACCHARIDE

Abstract

Detection and quantitation of lipopolysaccharide (LPS) are important for quality assurance of pharmaceutical, biopharmaceutical products and for several research and industrial aspects. The widely available assays for LPS detection are the normal, or chromogenic, and the synthetic LAL (Limulus amebocyte lysate). Unfortunately, both assays are expensive and could not distinguish between different types of bacterial LPS, while LPS silver nitrate staining requires more than 20 hr and can only detect 5 ng LPS. The current modified protocol was able to detect less than 0.5 ng LPS in a polyacrylamide–urea gel. The procedure is rapid, inexpensive, and reproducible. It depends on introducing two modifications to improve the staining sensitivity in sample buffer and staining procedure. The results revealed that excluding the reducing agent sucrose to use instead glycerol, and replacing SDS with DOC, as well as incorporation of 4 M urea in stacking and separating gels, increased the sensitivity up to 150 pg. In summary, the gels were fixed, carbohydrates moieties were oxidized to create active aldehydes, and the gels were silver stained using non-ammoniacal silver nitrate.

Keywords:

• ammoniacal and aqueous silver nitrate
• lipopolysaccharides
• sample buffer
• sensitivity
• separation

Notes

The author's current address is Elrashdy M. Redwan, Biological Science Dept., Faculty of Science, P.O. Box 80203, King Abdul Aziz University, Jeddah 21589, Saudi Arabia.