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本文对硫酸铵盐析及DEAE纤维素柱层析相结合提取小鼠lgG的方法进行了一些改进,在柱层析时采用增加磷酸缓冲液(P B液)离子浓度进行洗脱,获得较纯的lgG,并采用小鼠lgG干重制定标准曲线进行蛋白定量。方法和结果如下: 取20ml正常小鼠血清分别35%饱和硫酸铵盐析1次及40%饱和硫酸铵盐析2次后,取沉淀物透析至无NH~+进行DEAE纤维素柱层析。把盐析后lgG粗提物稀释后样品(约7ml)加入层析柱至样品全部进入柱体后关闭下口2～4小时。开放下口先用0.01M pH7.1 PB液洗脱,层析图谱出现一小峰,曲线平稳后改换PBS液(每1000ml PB液中加8.5g氯化钠)洗脱,出现一高峰。收集高峰洗脱样品浓缩进行紫外吸收值测定。取2ml lgG样品冷冻干燥称重,然后以PB液溶解,稀释不同浓度,以PBS液做对照,测紫外吸收值,绘制小鼠血清lgG含量标准曲线。用
In this paper, some methods of extracting mouse lgG by ammonium sulfate salting and DEAE cellulose column chromatography were improved. In column chromatography, elution was performed by increasing the concentration of phosphate buffer (PB solution) to obtain purer IgG. lgG, and use mouse lgG dry weight to establish a standard curve for protein quantification. Methods and results are as follows: Take 20ml normal mouse serum 35% saturated ammonium sulfate salting out 1 times and 40% saturated ammonium sulfate salting out twice, then take the precipitate to dialyzed to the absence of NH~+ for DEAE cellulose column chromatography. After salting out, the diluted sample of lgG crude extract (about 7 ml) was added to the column until the sample entered the column and the lower mouth was closed for 2 to 4 hours. Under the open mouth first eluted with 0.01M pH7.1 PB solution, a small peak appeared in the chromatogram. After the curve was stable, the PBS solution was changed (add 8.5g of sodium chloride per 1000ml of PB solution) to elute. The peak elution sample was collected and concentrated for UV absorption determination. 2ml lgG samples were lyophilized and weighed, then dissolved in PB solution and diluted to different concentrations. The PBS solution was used as a control to measure the UV absorbance and a standard curve of mouse serum lgG was drawn. use