ProteinExpressionandPurificationProtocol
Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)a) Take competent cell stock aliquot (about 100 mL) out of -80oC freezer. Place on ice to thaw about 5 minutes.b) Take one mL of plasmid DNA (usually a pRSET B vector with your protein cloned inside DNA seq of pRSET B) and add to the thawed com......阅读全文
Protein－Expression－and－Purification－Protocol
Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)a) T
Protein－A－Purification－of－Antibody
1. Reagents(1) Affi-gel Protein-A Agarose (BioRad #153-6153)(2) MAPS II Binding Buffer (BioRad # 153-6161)(3) 0.314 g/ml diH2O(4) MAPS II Elution Buff
Protein－purification;－actin
Protein purification; actin Overview ACTINThe most abundant muscle and non-muscle cytoskeletal protein. MW 42 kDa, 374/375 amino acids; various
Protein－G－Purification－of－Antibodies
1. Reagent and Materials(1) Hi-Trap Protein G Column (Pharmacia Biotech #17-0404-01)(2) 20 mM Sodium Phosphate Buffer, pH 7.01.084 g NaH2PO4, anhydrou
Antibody－Purification－using－Protein－A,－Protein－G,－or－Protein－L－Agarose
实验概要This protocol is designed as a quick purification method for antibodies from mammalian sera, ascites, and cell culture supernatants. It should
Antibody－Purification－using－Protein－A,－Protein－G,－or－Protein－L－Agarose
实验概要This protocol is designed as a quick purification method for antibodies from mammalian sera, ascites, and cell culture supernatants主要试剂 Protein
Measurement－of－Green－Fluorescent－Protein－Expression
ReagentsCells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as control.Hoechst 33342 s
Protocol－for－Protein－Extraction－for－proteomics
Protocol for Protein Extraction10 % w/v TCA/ acetone/ 0.07 % v/v -MercaptoethanolPlant cells are rich in compounds that interfere with the 2DE separat
troponin蛋白纯化－Protein－purification:－troponins
Overview TROPONINS The calcium-dependent regulatory protein complex located on the thin actin filaments of muscle comprises of TnC (17.8 kDa), TnI (2
Purification－of－Antiserum－or－Ascites－by－Protein－A/G－Chromatography
1、Required Materials and Equipment(1) Protein A or G agarose gel column (10 ml or 5 ml of packed beads; see guidelines below for choice of protein A o
蛋白质相互作用
Interaction Trap/Trap Two-Hybrid System· Yeast Two-Hybrid System (Finley Lab)This is one of the most comprehensive and detailed guide to yeast
蛋白表达
Protein Construct Expression and Purification Procedures (Gimila's Lab) Protein Expression (Mark's Lab) · Purification of GST Fused
Purification－of－Kar3－Motor－Domain－Protein
Purification of Kar3 Motor Domain ProteinMaterialsInduced cells (2 - 5 g pellet of pET/Kar3 in BL31(DE3)pLysS host cells) (See note #1)HEM buffer =10
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
酵母双杂交系统
· Yeast Two-Hybrid System (Finley Lab)This is one of the most comprehensive and detailed guide to yeast two-hybrid system technique with intro
SDSPAGE检测蛋白表达(protein－expression)
一、材料与仪器30%丙烯酰胺溶液；1.5mol/L Tris-HCl分离胶缓冲液，PH8.8；1.0mol/L Tris-HCl浓缩胶缓冲液，PH6.8；电泳缓冲液，PH8.3；10%SDS溶液；10%过硫酸铵溶液；样品处理液；染色液；脱色液；电泳玻璃板，电泳电源架，电泳槽，电泳仪等；蛋白Mar
GST融合蛋白的准备
Preparation of Glutathione-S-Transferase (GST) Fusion ProteinsMargret B. Einarson and Elena N. Pugacheva Foxx Chase Cancer Center, Philadelphia, PA 19
蛋白质纯化(protein－purification)实用技术2
7．密度多数蛋白质的密度在1.3~1.4g/cm3之间，分级分离蛋白质时一般不常用此性质，不过对含有大量磷酸盐或脂质的蛋白质与一般蛋白质在密度上明显不同，可用密度梯度法离心与大部分蛋白质分离。8．基因工程构建的纯化标记通过改变cDNA在被表达的蛋白的氨基端或羧基端加入少许几个额外氨基酸，这个加入的标
蛋白质纯化(protein－purification)实用技术3
10．非极性基团之间作用力溶质分子中的非极性基团与非极性固定相间的相互作用力（非选择性分散力或伦敦力）大小与溶质分子极性基团与流动力相中极性分子在相反方向上相互作用力的差异进行分离。因其流动相中的置换剂是极性小于水的有机溶剂（如甲醇、乙腈、四氢呋喃等），这些有机溶剂可能使许多蛋白质分子产生不可逆的变
蛋白质纯化(protein－purification)实用技术1
研究的最后还是要看基因表达产物,无论是用于检测还是用于棉衣保护,都需要将表达出的蛋白质分离和纯化,然而蛋白质性质各异,故纯化方法不同,现共享一些基本的纯化方法,以飨读者:蛋白质的一级、二级、三级和四级结构决定了它的物理、化学、生物化学、物理化学和生物学性质，综述了不同蛋白质之间的性质存在差异或者改变
Jacobs:Protocol－Total－Protein－Isolation－Using－RIPA－Lysis－Buffer
MaterialsRIPA buffer (RIPA buffer enables the extraction of cytoplasmic, membrane and nuclear proteins and is compatible with many applications, inclu
抗体纯化
Antibody PurificatioinPurification of IgG Using Protein A- or Protein G-Agarose (KPL) Purifying Antibodies (Perkin-Elmer)Precipitation MethodsProtein
GST融合蛋白（GST－fusion－protein－purification）的表达与纯化
原理GST 纯化系统是利用GST （glutathione-S-transferase ）融合蛋白与固定的谷胱甘肽（GSH）通过硫键共价亲和，通过GSH交换洗脱的原理来进行纯化 。1ml树脂大约可结合5-8 mg融合蛋白，并可反复使用数次。试剂u IPTG（异丙基硫代-β-D-半乳糖苷） 2
单克隆抗体
· Tips and hints for the storage of antibodies (Synaptic Systems)· Purification of IgG Using Protein A- or Protein G-Agarose(KPL)·
Expression－Protocol－in－M9－Minimal－Media－via－T7－Promoter
The following protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 min
Expression－Protocol－in－M9－Minimal－Media－via－T7－Promoter
Expression Protocol in M9 Minimal Media via T7 PromoterThe following protocol has been used successfully to 15N or 13C/15N label our proteins using ou
Doubletag在蛋白纯化(Protein－Purification)过程中的应用
Protein表达是一个非常复杂的过程，因此很难预测表达的蛋白是否是可溶性的、包涵体形式还是部分降解的。为了预防各种可能的困难条件，在重组蛋白上导入两种Tag可以提供获得高纯度均质蛋白的灵活性。 蛋白含有两种不同亲和纯化Tag的重要原因： 纯化全长的蛋白 获得最高纯化的蛋白 适合变性
DNA微序列技术
· Protocols for Making Drosophila Arrays (Stanford U.)Detailed protocol for making arrays including PCR Amplification of cDNAs for Printing,
蛋白质磷酸化
Tyrosine Kinase Assay Using Synthetic Peptides (T. Miller)Small synthetic peptide substrates are especially well suited for applications such as assay
Expression－L19－using－Pichia－pastoris
Pichia pastoris is a methylotropic yeast used to express high amounts of protein. Secreted expression is achieved with a cleavalable faktor. To yield