蛋白分离纯化

GST Protein Prep.Ver.2 1)Grow 50ml of culture in LB or TB antibiotic o/n at 37℃ shaker. 2)Dilute culture in LB or TB antibiotic 1:10 3)Grow 3hrs at 37℃. 4)Induce culture by adding 0.4 mM IPTG final co ...

Screen of GST-Fusion Protein Expression (pGEX system by Amersham: for check clones for expression of the desired fusion protein prior to large-scale purification) Pick several colonies of E.coli trans ...

Buffers: Lysis Buffer (1 liter) 50 mM NaH2PO4 6.90g NaH2PO4.H2O (MW 137.99g/mol ) 300 mM NaCl(up to 2M)17.54gNaCl(MW 58.44)or 60ml 5M 10 mM Imidazole (up to 100mM)0.68g (MW 68.08 ) 10 mM BME (up to 20 ...

Horvath and RiezmanYeast1994; Gottschling Lab Sample Buffer: 10 ml 0.06M Tris-HClpH 6.8 ...

SDS extraction followed by acetone precipitation – simple extraction protocol that does not require phenol.Recommended start protocol for whole tissue extractions. 1.Grind 1 g of fresh tissue to ...

Buffer A Buffer B &nb ...

Purification of TFIIA from E.coli Linda WarfieldHahn Lab2003 Volumes given are for 2L of cells for each subunit (Toa1 and Toa2). Toa1 and Toa2 subunits are expressed separately in cultures of OD0.6 fo ...

To concentrate proteins for analysis by SDS PAGE: If a small amount of protein is to be precipitated (less than a few micrograms)add Insulin as a carrier protein (10 micrograms of Sigma insulinI-5500p ...

Summary WCE Extracts are prepared from strain SHY282 (TFC4-Flag-EE (HpaI)).TFIIIC complex is first purified by flag affinity: TFC4 contains a single copy of the flag epitope.The flag purified TFIIIC i ...

Horvath and RiezmanYeast1994; Gottschling Lab Sample Buffer: 10 ml 0.06M Tris-HClpH 6.8 0.6 ml 1M Tris 6.8 10% (v/v)glycerol 2 ml 50% glycerol 2% (w/v)SDS 2 ml 10% SDS 5% (v/v)2-mercaptoethanol 0. ...

Adapted from YaffeM.P.and SchatzG.PNAS1984814819-4823. 1.Grow 25mls yeast cells to 5x10E6. 2.Sequentially spin down cells in a 15 ml polypro tube. 3.Wash cells with 1ml ice cold ddH2Otransferrring to ...

Solutions Elution Buffer 50 mM Tris 7.5 5 ml 1M Tris pH 7.5 0.1% SDS 0.1 g SDS 0.1 mg/ml BSA 1.0 mg BSA 0.25 mM EDTA 50 ml 0.5M EDTA 2.5% glycerol 2.5 ml glycerol up to 100 ml with Q For every 10 ml a ...

TGEK Base 50 mM Tris 10% vol Glycerol 1 mM EDTA 100 mM KCl (add for std lysis buffer): PMSF Benzamidine Leupeptin Aprotinin 0.5% NP40 (add for solubilization buffer) 1% NP40 0.1% Triton X100 0.1% SDS ...

Preparation of Brain Membrane Fractions for Western Blot Analysis All the procedures should be done at 4ºC using precooled reagents. For rat samples immediately remove brain from the cranium into ...

Reagents / Solutions Lysis Buffer: 10ml 10% Sodium dodecyl sulphate (SDS) 10ml Glycerol 10ml b-mercaptoethanol 8ml 0.5M Tris pH6.8 1ml 0.1% bromophenol blue 51ml H2O Protocol Spin down 106 cells and ...

Solutions: Buffer A (Hypotonic Buffer): 1L 10 mM HEPES pH 7.9 10 ml 1M HEPESpH 7.9 1.5 mM MgCl2 1.5 ml 1M MgCl2 10 mM KCl3.33 ml 3M KCl 0.5 mM DTT 500 µl 1M DTT 0.2 mM PMSF 1 ml 0.2 M PMSF Buffe ...

（一）双缩脲测定法 1．原理 蛋白质中的肽键有双缩脲反应，在碱性溶液中与二价铜离子形成蓝紫色的络合物，在一定的范围内，颜色的深浅与蛋白质的含量成正比。此法特异性强，游离的氨基酸、小肽和核酸均不产生这种反应，但此法不够敏感，仅能测出毫克水平。 2．试剂配制 硫酸铜（CuSO4·5H2O） 1.50g 酒石酸钾钠 5.00g H2O 500.0ml 10%氢氧化钠（不含硫酸钠）300m ...

Grow a 2 ml culture24 hr.at 30℃ in selective media When culture is readyuse it to inoculate about 55 ml (50 ml plus 5 for O.D.600 readings)of selective media.Grow to mid-logarithmic phase (O.D.600 is ...

随着分子生物学的发展，越来越多的科研人员熟练掌握了分子生物学的各种试验技术，并研制成套试剂盒，使基因克隆表达变得越来越容易。但分子生物学的上游工作往往并非是最终目的，分子克隆与表达的关键是要拿到纯的表达产物，以研究其生物学作用，或者大量生产出可用于疾病治疗的生物制品。相对与上游工作来说，分子克隆的下游工作显得更难，蛋白纯化工作非常复杂，除了要保证纯度外，蛋白产品还必须保持其生物学活性。纯化工艺必须 ...

到目前为止，蛋白质结构解析的方法主要是两种，x射线衍射和NMR。近年来还出现了一种新的方法，叫做Electron Microscopy。其中X射线的方法产生的更早，也更加的成熟，解析的数量也更多，我们知道，第一个解析的蛋白的结构，就是用x晶体衍射的方法解析的。而NMR方法则是在90年代才成熟并发展起来的。这两种方法各有优点和缺点。 首先来说一下，这两种方法的一般的步骤和各自的优点和缺点。电子显微镜 ...