色谱法
鲎试剂
化学
显色的
尿素
聚丙烯酰胺
染色
硝酸银
脂多糖
分析灵敏度
尿素酶
银染
甘油
生物化学
分子生物学
生物
核化学
医学
遗传学
替代医学
病理
高分子化学
内分泌学
标识
DOI:10.1080/10826068.2011.586081
摘要
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.
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