Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.
Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.
复制标题
肌动蛋白 N 末端片段的免疫化学探测：聚合反应。
DOI：
10.1021/bi00465a024
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Reisler,E
中科院分区：
文献类型：
--
作者：
DasGupta,G;White,J;Phillips,M;Bulinski,JC;Reisler,E
材料与方法在所有实验中使用蒸馏水和Millipore过滤水以及分析级试剂。木瓜蛋白酶、碱性磷酸酶缀合的山羊抗兔IgG、溴化氰活化的琼脂糖、碱性磷酸酶底物、弗氏佐剂、TLCK处理的α-胰凝乳蛋白酶和ATP购自Sigma Chemical Co. (St. Louis，MO）。N-（1-芘基）碘乙酰胺获自Molecular Probes（联合城，OR）。用于兔免疫的合成肽Ac-Asp-Glu-Asp-Glu-Thr-Thr-Ala-Tyr购自加州大学圣地亚哥分校的定制肽合成设施。~（14）C标记肽的比活性为35 cpm/nmol。钥孔血蓝蛋白来自Calbiochem（拉霍亚，CA）。Bradford蛋白测定溶液和蛋白A-琼脂糖来自Bio-Rad（里士满，CA）。ELISA板（Dynatech Immulon I）购自FisherScientific Co.Preparation of Proteins.通过Spudich和Watt（1971）的方法，在G-肌动蛋白缓冲液（0.5 mM/3-巯基乙醇、0.2 mM ATP、0.2 mM CaCl 2和5 mM Tris，pH 7.6）中制备骨骼肌肌动蛋白。按照MacLean-Fletcher和Pollard（1980）的描述获得凝胶过滤的肌动蛋白。按照戈弗雷和哈灵顿（1970）的描述制备肌球蛋白。亚片段1
Materials and MethodsReagents. Distilled and Millipore-filtered water and ana-lytical-grade reagents were used in all experiments. Papain, alkaline phosphatase conjugated goat anti-rabbit IgG, cyanogen bromide activated Sepharose, alkaline phosphatase substrate, Freund’s adjuvent, TLCK-treated a-chymotrypsin, and ATP were purchased from Sigma Chemical Co.(St. Louis, MO). 7V-(l-Pyrenyl) iodoacetamide was obtained from Molecular Probes (Junction City, OR). The synthetic peptide Ac-Asp-Glu-Asp-Glu-Thr-Thr-Ala-Tyr used for immunization of rabbits was purchased from the custom peptide synthesis facility at the Universityof California, San Diego. The specific activity of the l4C-labeled peptide was 35 cpm/nmol. Keyhole limpet hemocyanin was from Calbiochem (La Jolla, CA). Bradford protein assay solution and protein A-agarose were from Bio-Rad (Richmond, CA). ELISA plates (Dynatech Immulon I) were purchased from FisherScientific Co.Preparation of Proteins. Skeletal muscle actin was prepared in G-actin buffer (0.5 mM/3-mercaptoethanol, 0.2 mM ATP, 0.2 mM CaCl2, and 5 mM Tris, pH 7.6) by the procedure of Spudich and Watt (1971). Gel-filtered actin was obtained as described by MacLean-Fletcher and Pollard (1980). Myosin was prepared as described by Godfrey and Harrington (1970). Subfragment 1