回肠消化物乙醇沉淀物分析：一种测定黏蛋白方法的评估
Analysis of an ethanol precipitate from ileal digesta: evaluation of a method to determine mucin.
作者信息
Miner-Williams Warren M, Moughan Paul J, Fuller Malcolm F
机构信息
Riddet Institute, Massey University, Palmerston North, New Zealand.
出版信息
Sci Rep. 2013 Nov 6;3:3145. doi: 10.1038/srep03145.
The precipitation of mucin using high concentrations of ethanol has been used by many researchers while others have questioned the validity of the technique. In this study, analysis of an ethanol precipitate, from the soluble fraction of ileal digesta from pigs was undertaken using molecular weight profiling and polyacrylamide gel electrophoresis. The precipitate contained 201 mg·g⁻¹ protein, 87% of which had a molecular weight >20 KDa. Polyacrylamide gel electrophoresis stained with Coomassie blue and periodic acid/Schiff, revealed that most glycoprotein had a molecular weight between 37-100 KDa. The molecular weight of glycoprotein in the precipitate was therefore lower than that of intact mucin. These observations indicated that the glycoprotein in the ethanol precipitate was significantly degraded. The large amount of protein and carbohydrate in the supernatant from ethanol precipitation indicated that the precipitation of glycoprotein was incomplete. As a method for determining the concentration of mucin in digesta, ethanol precipitation is unreliable.
许多研究人员使用高浓度乙醇沉淀黏蛋白，而其他一些人则质疑该技术的有效性。在本研究中，利用分子量分析和聚丙烯酰胺凝胶电泳对猪回肠消化物可溶部分的乙醇沉淀物进行了分析。该沉淀物含有201 mg·g⁻¹蛋白质，其中87%的分子量>20 kDa。用考马斯亮蓝和过碘酸/希夫试剂染色的聚丙烯酰胺凝胶电泳显示，大多数糖蛋白的分子量在37 - 100 kDa之间。因此，沉淀物中糖蛋白的分子量低于完整黏蛋白的分子量。这些观察结果表明，乙醇沉淀物中的糖蛋白已被显著降解。乙醇沉淀上清液中大量的蛋白质和碳水化合物表明糖蛋白沉淀不完全。作为一种测定消化物中黏蛋白浓度的方法，乙醇沉淀法并不可靠。
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