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
“OneStep－Digestion”快速酶解试剂盒
快速酶解试剂盒——更快、更准确地进行蛋白质表征和研究轻松简单• 流程简单，一步完成样品处理• 变性还原烷基化酶解全流程只需半小时结果可靠• 降低样品处理过程引入的蛋白修饰• 提高蛋白酶解重现性• 增强数据可信度“One-Step Digestion”快速酶解试
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
Competitive－RTPCR－Strategy－for－Quantitative－Evaluation－5
3. Characterization of the method precision and repeatability.a. Ensemble PCRs in the same conditions established before using quantities of target in
DNA－Purification－from－Agarose－Gels
1. Separate DNA fragments in an agarose gel cast with 0.5 mg/mL Ethidium bromide. Locate bands with a hand-held long-wave UV lamp.2. Slice the gel wit
Isolation－of－Cell－Wall－Proteins－from－Medicago－sativa－Stems
Plant cell walls are highly dynamic and chemically active components of plant cells. Cell walls consist primarily of polysaccharides, with protein
目标蛋白(target－proteins)粗提方法1
狭义来讲，提取指制备物与细胞固体成分或其它结合成分的分离，由固相转入液相或从细胞内生理状态转入外界特定溶液环境的过程，即分离前期被提取物从破碎的细胞中释放出来的过程。如果被提取物程固相或与固体结合，提取时由固相转入液相，常称为固液萃取；如果被提取物已经呈液相存在，提取时由一液相转入转入另一互不相溶的
目标蛋白(target－proteins)粗提方法3
3）pH值 一般在被提取蛋白质的等电点的两侧，碱性蛋白用稀酸提取、酸性蛋白用稀碱提取。在某些情况下，为了破坏所分离的蛋白质与其他杂质的静电结合，选择偏酸（pH3-6）或偏碱（pH10-11），可以使离子键破坏而获得单一的蛋白成分。4）温度 提取时通常要求低温操作。只有对某些耐高温的蛋白质或酶（如胃蛋
目标蛋白(target－proteins)粗提方法2
4）去垢剂去垢剂(表面活性剂)是一类即具有亲水基又具有疏水基的物质，一般具有乳化、分散、和增溶作用，可分阴离子、阳离子和中性去垢剂等多种类型，中性去垢剂在蛋白提取钟应用的较多。A．中性去垢剂 又称非离子表面活性剂，对蛋白质的变性作用影响较少，宜于蛋白质或酶提取之用。一般市售中性去垢剂有聚乙二醇类
目标蛋白(target－proteins)粗提方法4
第四节 提取时的保护性措施提取一些具有生理活性的物质时，除了考虑被提取物溶解度外，还应考虑被提取物活性的保护。对于一些生物大分子如蛋白质、酶和核酸来说，主要措施有如下几点；1．采用缓冲系统 防止提取过程中某些酸碱基团的解离，造成溶液中PH值变化幅度过大，导致某些活性物质的变性、或由于PH变化影响
Phosphoamino－acid－analysis－of－nonTCA－precipitable－proteins
Phosphoamino acid analysis of non-TCA precipitable proteins1. Cut out gel slice, incubate 60 min with 1 ml 5.7 M HCl @ 110oC in 13 x 100 mm screw cap
Preparation－of－Bacterial－Proteins－for－Analysis－by－2DPAGE
The following protocol has been developed for preparing soluble bacterial proteins in a form suitable for analysis by 2D-PAGE. The procedure was princ
RNAi实验中双链短RNA（dsRNA）制备过程
RNAi 实验中双链短RNA（dsRNA）制备过程，本实验方法来自于加州大学Jim教授实验，很权威！Procedure for the Generation of dsRNA for use in RNAi1. Design polymerase chain reaction (PCR ) prim
Size－and－Shape－of－Protein－Molecules3
The Kirkwood/Bloomfield CalculationThe understanding of how protein shape affects hydrodynamics is elegantly extended by an analysis originally develo
Southern－blot实验方法和操作步骤
第一天 (restriction enzyme digestion)1.取待測的genomic DNA，用二次水稀釋10倍(30μl的DNA 加270μlddH2O→ 300μl)2.換算genomic DNA濃度(OD260)，再換算取10μg所需的體積。3.取10μg DNA，用限制酵素EcoR
缓冲液梯度聚丙烯酰胺凝胶的概念
中文名称缓冲液梯度聚丙烯酰胺凝胶英文名称buffer-gradient polyacrylamide gel定 义凝胶缓冲液成分有pH、离子强度或盐种类等连续或不连续梯度变化的聚丙烯酰胺凝胶。旨在提高凝胶电泳分离的范围和分辨力。应用学科生物化学与分子生物学（一级学科），方法与技术（二级学科）
缓冲液梯度聚丙烯酰胺凝胶的定义和用途
中文名称缓冲液梯度聚丙烯酰胺凝胶英文名称buffer-gradient polyacrylamide gel定 义凝胶缓冲液成分有pH、离子强度或盐种类等连续或不连续梯度变化的聚丙烯酰胺凝胶。旨在提高凝胶电泳分离的范围和分辨力。应用学科生物化学与分子生物学（一级学科），方法与技术（二级学科）
SAPK/Jun－kinase－assays
Preparation of cell lysate:1. For cells in suspension, grow in DMEM with 10% FCS and then the day before you do the experiment spin the cells down (1
The－ribonuclease－protection－assay－(RPA)
The ribonuclease protection assay (RPA) is a highly sensitive and specific method for the detection of mRNA species. The assay was made possible by th
Size－and－Shape－of－Protein－Molecules5
Hydrodynamic Analysis and EM Applied to Large Multisubunit ComplexesThe text box above showed the application of the Siegel–Monte analysis to SMC prot
Size－and－Shape－of－Protein－Molecules5
Hydrodynamic Analysis and EM Applied to Large Multisubunit ComplexesThe text box above showed the application of the Siegel–Monte analysis to SMC prot
Histone－blotting－protocol
实验概要 Western blot detection of histone proteins. 实验步骤 The following protocol refers to the western blot detection of histone proteins derived from p
Histone－blotting－pr...
实验概要The method provides a procedure and tips for detecting histone proteins.The protocol refers to the western blot detection of histone proteins d
引物延伸反应
Primer extension analysis is used to determine the location and quantitate the amount of the 5´-end of specific RNAs. An end-labeled oligonucleotide i
RNA实验方法
Solublization of RNA in Formamidecontributed by James McCaughern-Carucci, Yale UniversityResuspending RNA in Formamide (as reported by Chomczynski et
RapiGest－SF－试剂：促进溶液中蛋白酶解的有利工具（三）
与其他的蛋白酶合用 我们测试了 RapiGest SF 与多种蛋白酶的适配性，如 Asp-N, Lys-C 与 Glu-C 。在酶解前使用 RapiGest SF 变性蛋白获得了有效的消解结果。蛋白去糖基化的用途 RapiGest SF 也用于测试其它酶，如 PNGase F ，该酶用于酶切糖蛋白
Keratocyte－isolation－(corneal－fibroblasts)
Corneal keratocytes (corneal fibroblasts) that this corneal layer is specialized fibroblasts residing in the stroma, representing about 85-90% of
PCR实验指导与常见问题分析5
MgCl2 concentrationRelationship between MgCl2 and dNTP concentrationdNTP concentrations of about 200µM each are usually recommended for the Taq polyme
EGel®－CloneWell－Agarose－Gels
实验概要Instructions are provided below for using the E-Gel®CloneWell pre-cast agarose gels with the E-Gel® iBase™ Power System. For detailed instructio
琼脂扩散试验（gel－diffusion－test）
可溶性抗原与相应抗体在半固体琼脂凝胶内扩散，二者相遇，在比例合适处形成白色沉淀。基本类型有单向扩散和双向扩散两种。 （一）双向琼脂扩散试验（Double immunodiffusion test） 抗原和抗体加到琼脂板上相对应的孔中，两者各自向四周扩散，如两者相对应，浓度比例合适，则经一定时间后，在
HP－Tissue－DNA－Maxi－Protocol
实验概要The E.Z.N.A.® HP Tissue DNA Maxi Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from up to 2 grams of tissue samples.