Rider M H, Puype M, Van Damme J, Gevaert K, De Boeck S, D'Alayer J, Rasmussen H H, Celis J E, Vanderkerchove J
Hormone and Metabolic Research Unit, University of Louvain Medical School, Brussels, Belgium.
Eur J Biochem. 1995 May 15;230(1):258-65. doi: 10.1111/j.1432-1033.1995.0258i.x.
An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting.
本文描述了一种基于琼脂糖的浓缩凝胶系统，用于洗脱和浓缩先前在一维或二维凝胶中纯化的蛋白质。使用该技术，蛋白质可从约1毫升浓缩至小至10微升的体积。蛋白质从琼脂糖凝胶中熔化出来后，可用蛋白酶进行消化，产生与溶液消化中观察到的类似的肽图谱。从加载到初次分离凝胶上的蛋白质量到HPLC后片段收集所计算出的总肽回收率，至少是相同量蛋白质在游离溶液中消化所获得肽产量的70%。这些结果通常是用50皮摩尔量（指最初加载到初次凝胶上的蛋白质量）获得的。蛋白质还可以通过微量测序和在线电喷雾质谱联用进行分析，从而通过肽质量指纹图谱对其进行鉴定。
