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[求助]
急求：怎样确定蛋白和肽有没有结合？
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| 怎样确定一个4000多分子量的蛋白，是否与700多分子量的肽结合了？ |
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lzjessie_wlf
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【答案】应助回帖
soundhorizo: ACI+1, 感谢您的专业应助 ^^ 常来哦~ 2012-07-12 23:36:21
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还有就是使用Isothermal titration calorimetry. 使用那种用量少的 microIsothermal titration calorimetry. http://www.microcal.com/products/itc/itc200.asp iTC200 Isothermal Titration Calorimetry The iTC200 system is the most sensitive isothermal titration calorimeter available. It is designed to address the needs of today’s life science researchers – particularly those engaged in drug discovery and development. With its reduced size and associated sample quantity requirements, the iTC200 is significantly faster than previous models. Designed for ease-of-use, all functions of the iTC200 are operated through software to facilitate fast and accurate analyses. The iTC200 utilizes a 200 μL cell versus the 1.4 mL cell in previous models. Applications include: Characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules. Lead optimization. Enzyme kinetics. Assessment of the effect of molecular structure changes on binding mechanisms. Assessment of biological activitiy. Why iTC200? More than just affinities: Simultaneous determination of all binding parameters in a single experiment - information unobtainable from more limited binding assays. Directly measure sub-millimolar to nanomolar binding constants (102 to 109 M-1). Measure nanomolar to picomolar binding constants (109 to 1012 M-1) using the competitive binding technique. Application versatility: Investigate any biomolecular interaction with high sensitivity. The sample cell is 200 μL. As little as 10 μg of protein can be used. True in-solution technique: No labeling or immobilization required. No buffer restrictions. Easily handles turbid solutions. Easy to use: Includes user friendly experimental design wizards and easy filling and cleaning procedures. Fast time to first result: With fast equilibration times, up to two runs per hour can be easily accomplished. Requires no assay development or dedicated user to achieve high quality results. Automatable: iTC200 can be upgraded to a fully automated version with a sample throughput of at least 75 samples per day with a capacity to process as many as 384 samples unattended. At Rest Sample Loading Titration Washing iTC200 features: Active cell volume ~200 μL. Non-reactive Hastelloy® cells for excellent chemical resistance. Fixed-in-place cells for reproducible ultrasensitive performance with low maintenance. Precision liquid delivery system for accurate and reproducible injections User-selectable mixing speeds to match sample conditions. Three user selectable response times (U.S. Patent number 5,967,659) for application versatility. Operating temperature range of 2oC to 80oC. Peltier controlled for rapid temperature equilibration. Includes the iTC200 Washing Module for easy cell and syringe filling and cleaning. |
6楼2012-07-12 22:53:40
houqian387
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2楼2012-07-11 17:50:35
lzjessie_wlf
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【答案】应助回帖
★ ★ ★ ★ ★
感谢参与，应助指数 +1
soundhorizo: 金币+5, 感谢应助 ^^ 2012-07-11 21:58:54
感谢参与，应助指数 +1
soundhorizo: 金币+5, 感谢应助 ^^ 2012-07-11 21:58:54
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简单的化，直接用MS检测。 配置不同ratio的蛋白和肽，使用MALDI和ESI检测。 如果能检测到两个分子量之和，那么判断有可能结合了。 同时（或然后）做对比实验，比如配置相同的溶液，分别不含蛋白或者肽。 尤其是分子量小的肽，以免肽自身在某个浓度的溶液中形成multiplemer与其他小分子的complex，最后分子量与蛋白和肽的分子量之和类似。 MALDI可以直接检测到single charged的complex 峰。 ESI的化看机器的检测范围。如果检测范围小于4000,那么直接检测到的是multiple charged 的分子量，最后通过人工或机器deconvolution推到出single charged 的分子离子峰。 然后使用LC-MS 分离检测，如果LC-MS能够分离检测到，那么就可以做NMR。对特殊的hydrogen bond进行表征。 最后，还可以培养结合后蛋白和肽的共晶，使用X-ray检测。 总结，查相关类似文献。 |
3楼2012-07-11 21:44:41
4楼2012-07-12 10:12:21
lzjessie_wlf
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5楼2012-07-12 22:47:54
lzjessie_wlf
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7楼2012-07-12 22:58:39
8楼2012-07-13 09:34:11
lzjessie_wlf
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9楼2012-07-13 20:41:10
lzjessie_wlf
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llp15984810楼2012-07-14 01:56:25