蛋白质技术

0 128);"REAGENTSECL Western blotting kit (Amersham Life Science; cat# RPN2108): contains second antibodies for both mouse and rabbit substrate and milk blocker (the milk blocker is not normally used ...

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 ...

Trans-Bot SD Assembly 1. Prepare the transfer buffer. 2. Following electrophoresis equilibrate the gels in transfer buffer. Equilibration facilitates the removal of electrophoresis buffer salts ...

0 128);"Culture bacteria1. Grow glycerol stock A13 (TBP-GST in BL21) in 10mL LB and 10ul ampicillin (1:1000)2. Shake @ 37C ON 0 128);"Induce bacteria3. Adjust culture with LB until ON culture OD is ...

1. Treat cells. For PC12 or NIH 3T3 cells a moderately confluent 60 mm plate is used. 2. Aspirate media. Rinse cells with PBS aspirate. 3. Lyse cells in 200 uL of ...

SJF 4/001) Run gel (in 1:10 running/transfer buffer(10x) and H2O for a total of 1litre) at 150 volts until leading bromophenol blue band is nearing the bottom edge of the glass plates or according to ...

(All steps with agitation unless done O/N at 4oC) Rinse membrane once in TBS-TBlock the membrane with TBS-T/ 5% milk (or HIHS) for 30 min roomtemp (can be left at 4oC O/N here) Rinse membrane briefly ...

Steve Hahn last modified Mon Mar 22 1999Western Blot Probing by ECL1. After electroblotting of protein gel to immobilon membrane (See 0 128);"Novex NuPAGE method) mark the side of the membrane not in ...

0 128);"Buffers: 0 128);"TBS: 25 ml of 1 M Tris-7.5 30 ml of 5 M NaCl bring volume up to 1000 ml with ddwater 0 128);"TBS-T: TBS + 0.5 ml of Tween 20 0 128);"5X SDS-PAGE running buffer:&n ...

Semi-dry Transfer Apparatus Biorad Cat# 170-3940 or equivalent Power Supply 0 - 100 VDC (adj. current to 1 Amp) Immobilon-P transfer membrane 0.45 µm pore size; cut to same size as gel (Millip ...

Wash XCell box blotting module and trays with soap and water before proceeding. 0 128); font-weight: bold;"Reagents:Transfer buffer25 mM Tris HCl190 mM Glycine10% Methanol 0 0); text-decoration: unde ...

SDS电泳技术首先在1967年由Shapiro建立，1969年由Weber和Osborn进一步完善。当在样品介质和聚丙烯酰胺凝胶系统中加入SDS后，则蛋白质分子的电泳迁移率主要取决于它的分子量大小，其它因素可以忽略不计。 SDS是一种阴离子去污剂，它能破坏蛋白质分子之间以及其它物质分子之间的非共价键。在强还原剂如巯基乙醇或二硫苏糖醇的存在下，蛋白质分子内的二硫键被打开并解聚成多肽链。解聚后的蛋白质 ...

1. Load gel in apparatus as described by manufacturer. 2. Transfer proteins according to above ensuring that the NCM is toward the positive electrode. When using the Hoefer apparatus be sure to eit ...

This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system.

GeorgiaTimes"CARBONATE WESTERN TRANSFER SOLUTIONS Dunn Anal. Biochem. 1986: 157 GeorgiaTimes"1.5L GeorgiaTime ...

GeorgiaTimes"Blotting with BSA GeorgiaTimes"Blot in 5% mile/ 1X PBST (or 1X TBST) --60'/Rt GeorgiaTimes"Wash 2X for 5' in 1XPBST GeorgiaTimes"10Ab in 1% BSA/1X PBST --60'/Rt Geor ...