ProteinconcentrationofLaemmligelsamples
Protein concentration of Laemmli gel samplesTo 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin and wash with 10% TCA. Resuspend pellet in 100 µl 1M NaOH, add 900 µl water and assay 100 µl by Bradford method.......阅读全文
Protein－concentration－of－Laemmli－gel－samples
Protein concentration of Laemmli gel samplesTo 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin
Bradford－Protein－Concentration－Assay
Bradford Protein Concentration Assayversion 01/07/2001Abbreviations:mcg = microgramsmcL = microlitersBSA = bovine serum albuminO.D. = optical densityd
蛋白质定量
Quantitative Determination of Peptides by Sulfhydryl (-SH) Groups New (Contributed by David Van Horn, Dept. of Chemistry, UC Berkeley Greg Bulaj, Dept
Western－blotting样品准备－（二）
Sodium orthovanadate preparationAll steps to be performed in a fume hood. a. Prepare a 100 mM solution in double distilled water. b.
蛋白浓缩(protein－concentration)方法详解
1，透析袋浓缩法利用透析袋浓缩蛋白质溶液是应用最广的一种。将要浓缩的蛋白溶液放入透析袋（无透析袋可用玻璃纸代替），结扎，把高分子（6000－12000）聚合物如聚乙二醇（碳蜡）、聚乙烯吡咯、烷酮等或蔗糖撒在透析袋外即可。也可将吸水剂配成30%－40%浓度的溶液，将装有蛋白液的透析袋放入即可。吸水剂用
蛋白浓缩（protein－concentration）基本方法
蛋白浓缩方法基本有：丙酮沉淀法；免疫沉淀法；三氯醋酸沉淀法；硫酸铵沉淀法；（低温）有机溶剂沉淀法；聚乙二醇沉淀法；超滤法；透析法；离子交换层析和冷冻干燥法…… 1.丙酮沉淀法；三氯醋酸沉淀法 试验要求的仪器简单，但是常常导致蛋白质变性。 2.免疫沉淀法：得有特异性抗体！ 3.硫酸铵沉淀法
Western－blotting样品准备
实验概要Preparation of lysis buffers, protease and phosphatase inhibitors, lysate from cell culture, lysate from tissues, protein concentration, samples
Native－gel－electrophoresis（非变性电泳）
Native gel electrophoresis Under native PAGE conditions, polypeptides retain their higher-order structure and often retain enzymatic activity and inte
Bacic－Immunoprecipitation
实验概要 Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting,
Immunoprecipitation
实验概要In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex
Chromatin－Electrophoresis
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus CollegeExercise 10.4 - Chromatin ElectrophoresisLEVEL IIMaterials 14 M Urea6 M NaCl0.05
Immunoprecipitation
Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting, is rou
包涵体表达蛋白的纯化方法
Joseph SambrookPeter Maccallum Cancer Institute and The University of Melbourne, AustraliaDavid W. RussellUniversity of Texas Southwestern Medical Cen
Dephosphorylation－...
实验概要The method provides a protocol for removal of phosphate groups from proteins, before or after blotting. To demonstrate the specificity of an ant
Native－chromatin－immunoprecipitation－protocol
实验概要The method is a native chromatin immunoprecipitation protocol.主要试剂1. 10 x TBS 0.1 M Tris-HCl (pH 7.5) 1.5 M NaCl 30 mM CaCl2 20 mM MgCl2 50 mM Na
自然染色质免疫沉淀实验设计
1. The preparation of native chromatin from cultured human cells1.1.Cultured cells (e.g. HL-60 or lymphoblastoids) are grown to a density of approxima
Native－chromatin－immunoprecipitation－protocol
实验概要Native chromatin immunoprecipitation to query specific chromatin states of individual genes. 主要试剂10 x TBS 0.1 M Tris-HCl (pH 7.5) 1.5 M NaCl 30 mM
Verification－of－In－...
实验概要Environmental stresses often trigger rapid generation of reactive oxygen species (ROS) in plants. Excessive amount of ROS can cause damage to
同位素法测定底物磷酸化活性方法
实验概要Ideally, one would like to be able to directly phosphorylate substrates in an intact cell. This could potentially be performed by introducing AT
非变性胶蛋白电泳
Section 2.1Nondenaturing Polyacrylamide Gel Electrophoresis of ProteinsJohn M. Walker1. IntroductionSDS-PAGE (Section 2.2) is probably the most commo
美国实验室wetern方法
WESTERN BLOT PROTOCOLIn a Western blot, proteins that are separated on polyacrylamide gels on the basis of size are transferred to a membrane for dete
Bradford－protein－assay
Bradford protein assayConsiderations for useThe Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly
PCR实验指导与常见问题分析7
11. All products in my multiplex reaction are weak. How can I improve the yield?Decrease annealing time in small steps (2º C)Decrease extension temper
同位素法测定底物磷酸化活性方法－Phosphorylation－of－Substrates
Phosphorylation of SubstratesScott T. Eblen, N. Vinay Kumar, and Michael J. WeberDepartment of Microbiology and Cancer Center, University of Virginia
ChIP－using－plant－samples
实验概要 The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitati
Crosslinking－chromatin－immunoprecipitation－(XChIP)－protocol
实验概要ChIP is a powerful tool that allows the specific identification of proteins or histone modifications to regions of the genome. Chromatin is isol
Identification－of－a－Mutant－Kinase/ATP－Analog－Pair2
Cellular Transfection and Immunoprecipitation Before proceeding with the experiments outlined below, all kinase pocket mutants should be characterize
Lipoprotein－Analysis－Week－2:－Electrophoresis
Lipoprotein Analysis Week 2: Electrophoresis IntroductionSDS polyacrylamide gel electrophoresis (SDS PAGE) will be used to assess the purification pr
peptide－fingerprint－mapping
Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimension
Ingel－digestion－of－proteins－for－peptide－fingerprint－mapping
Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimension