Warlow R S, Gooley A, Rajasekariah P, Oszarac N, Walls R S
Immunology Department, Repatriation General Hospital, Sydney, N.S.W., Australia.
Electrophoresis. 1995 Jan;16(1):84-91. doi: 10.1002/elps.1150160115.
A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.
报道了一种从纯化蛋白质中获取N端和内部肽段氨基酸序列的制备方法。该方法能可靠地从每个纯化蛋白质或肽段中14至30个残基之间产生高保真信号，氨基酸分析时背景较低。该程序依赖于在制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）过程中对蛋白质进行原位染色，并利用微浓缩器将少量蛋白质反复浓缩到小的聚偏二氟乙烯（PVDF）圆盘上，直到吸附了足够的量以产生强测序信号。蛋白质洗脱和随后的吸附可以用染料进行目视监测，最终产物，即吸附了蛋白质或肽段的PVDF圆盘，可以直接插入自动氨基酸测序仪中。
