InGelDigestionofProteinsSeparatedbyPolyacrylamideGelElectrophoresis
1. Excision of protein bands (spots) from polyacrylamide gelsRinse the gloves you use with water to avoid traces of dust in your sample.Rinse the gel with water.Excise spots with clean pipette tip (f 2 mm) cutting as close to the edge of the spot as possible (to reduce the volume of ''background'' gel)Transfer gel spot into a well of a 96-microtitre plate (Costar # 3363 + 3092)2. Reduction and alkylat......阅读全文
酵母人工染色体
· Easy YAC Preparation Method (Andrew Davies,Shaw lab)· Screening YAC libraries (Donis Keller Lab)This is a method for screening YAC l
Assay－of－superoxide－dismutase－activity2
Cuvette holders in the sample chamber of the spectrophotometer were thermo-controlled at 25°C. For the blank test, 100 ml of 50 mM potassium phosphate
Gene－splicing－and－mutagenesis－by－PCRdriven－overlap－extension
实验概要 Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing.Initial
Transcription,－Translation－of－S35Radiolabelled－Protein－and－Binding－to－GST
Prepare the template by linearizing 25ug plasmid DNA at the 3'' end of the insert. Phenol / chloroform extract, ethanol / NaCl precipitate and
寡核苷酸的相关操作
In this section, you will find techniques related to oligonucleotides, such as oligo purification by acrylamide gel, annealing two oligos to make doub
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.
General－Cloning－Protocols
Large Scale Preps: (See Large scale plsasmid prep protocol for more details)Cultures: Inoculate a 5 mL LB/Amp (50 - 100 µg/mL) culture in early a.m. w
DNA克隆
DNA克隆（主要内容如下）· General Procedure· PCR Cloning· Subcloning· ET Cloning· Vector Preparation· Ligation Re
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
Pulse－Field－Electrophoresis
Manipulating and analyzing DNA are fundamentals in the field of molecular biology. Indeed, separating complex mixtures of DNA into different sized fra
SOUTHERN－BLOTTING
Materials:Whatman 3 mm Blotting Papernitrocellulose (Schleicher & Schuell, Amersham) or nylon membrane filter (Amersham).Paper towels (preferably C-fo
Construction－of－BAC－Libraries：Megabase－DNA－Isolation
Megabase DNA IsolationMegabase-size DNA isolation from plantsTo construct large insert DNA libraries in BAC and YAC vectors, methods must be developed
胞外基质
ECM Cell Attachment Assay (LTI)Cell Adherence Inhibition Assay (LTI)General protocol--Either monoclonal antibody or RGD peptide is added along with th
聚丙烯酰胺凝胶电泳简介
聚丙烯酰胺凝胶电泳（英语： polyacrylamide gel electrophoresis，简称PAGE） ，是以聚丙烯酰胺凝胶作为支持介质的一种常用电泳技术，用于分离蛋白质和寡核苷酸。 作用原理：聚丙烯酰胺凝胶为网状结构，具有分子筛效应。它有两种形式：非变性聚丙烯酰胺凝胶电泳（Nati
蛋白质提取和纯化
蛋白质提取和纯化（主要内容如下）Protein Extraction Protein PurificationProtein PrecipitationColumn PreparatioinQ & A Posted in the Method ForumProtein ExtractionWhole
A－Method－for－Assaying－Deubiquitinating－Enzymes2
Table 1: Hydrolysis of 125I-labeled Ub-PESTc by the purified YUH1.Specific activity againstDUBsCbz-LRGG-AMC125I-labeled Ub-PESTcYUH13.2 x 10-105.1 x 1
蛋白复合体直接酶消化法
Direct Enzymatic Digestion of Protein ComplexesSherry Niessen, Ian McLeod and John R. Yates IIIDepartment of Cell Biology, The Scripps Research Instit
细胞组分和细胞器——细胞骨架
Fixation and Immunofluorescence of the Cytoskeleton (Mitchison Lab) Recycling Tubulin (Mitchison Lab) Labeling Tubulin and Quantifying Labeling Stoi
Preparation－of－Plasmid－DNA－by－Alkaline－Lysis－with－SDS:－Maxipreparation
实验概要Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS.主要试剂Buffers and SolutionsAlkaline lysis solu
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
Gelelongation－assay－for－type－II－fatty－acid－synthesis
Gel-elongation assay for type II fatty acid synthesisSrinivas KodaliAndrew GalgociSheo Singh Dr.Jun Wang Dr., jun_wang2@merck.com, Merck Research Labo
凝胶延长分析二型脂肪酸合成的方法
Gel-elongation assay for type II fatty acid synthesisSrinivas KodaliAndrew GalgociSheo Singh Dr.Jun Wang Dr., jun_wang2@merck.com, Merck Research Labo
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
electrophoresis－of－DNA
Agarose Gel Electroporesis of DNA Making the gel: 1. Place casting platform with well former sideways in gel stand where you wish to pour
DNA－Electrophoresis
What is Electrophoresis?Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. DNA is a negatively
通过细胞受体代谢生物素化进行图像分析
Metabolic biotinylation of mammalian cell receptors for imagingBakhos A. Tannous , btannous@hms.harvard.edu, Massachusetts General Hospital and Harvar
Mitochondrial－DNA－Isolation－from－Somatic－Embryogenic－Cell－Cultures－of－Larix
Mitochondrial DNA is isolated by a modification of the methods described by Wilson and Chourey (1984) and Radetzky (1990). Cell cultures at four days
SSR－GEL－and－Silver－Staining－Protocol
I. EQUIPMENT:DNA sequencing unit (35 x 45 cm) & 2000V power supplyClampsLg. plastic trays (4), about 43 x 50 x 8 cm, and one lidTwo rocking platformsH
Synaptic－Proteins－at－the－Synaptic－Junction
The postsynaptic density (PSD) is a submembranous structure at the postsynaptic membrane mainly at the excitatory synapses. The neurotransmitter recep
Purification－of－GST－Fused－Proteins
Day 1Set up an overnight culture in 100 ml LMM broth or 100 ml terrific broth containing 100ul 100 mg/mlAmpDay 2Add 40-50 ml o/n culture to 1 lt terri