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| 查看: 1916 | 回復(fù): 10 | |||
| 本帖產(chǎn)生 1 個(gè) ACI ，點(diǎn)擊這里進(jìn)行查看 | |||
llp159848新蟲 (小有名氣)
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[求助]
急求：怎樣確定蛋白和肽有沒有結(jié)合？
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| 怎樣確定一個(gè)4000多分子量的蛋白，是否與700多分子量的肽結(jié)合了？ |
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lzjessie_wlf
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【答案】應(yīng)助回帖
soundhorizo: ACI+1, 感謝您的專業(yè)應(yīng)助 ^^ 常來哦~ 2012-07-12 23:36:21
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還有就是使用Isothermal titration calorimetry. 使用那種用量少的 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樓2012-07-12 22:53:40
houqian387
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2樓2012-07-11 17:50:35
lzjessie_wlf
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【答案】應(yīng)助回帖
★ ★ ★ ★ ★
感謝參與，應(yīng)助指數(shù) +1
soundhorizo: 金幣+5, 感謝應(yīng)助 ^^ 2012-07-11 21:58:54
感謝參與，應(yīng)助指數(shù) +1
soundhorizo: 金幣+5, 感謝應(yīng)助 ^^ 2012-07-11 21:58:54
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簡單的化，直接用MS檢測。 配置不同ratio的蛋白和肽，使用MALDI和ESI檢測。 如果能檢測到兩個(gè)分子量之和，那么判斷有可能結(jié)合了。 同時(shí)（或然后）做對比實(shí)驗(yàn)，比如配置相同的溶液，分別不含蛋白或者肽。 尤其是分子量小的肽，以免肽自身在某個(gè)濃度的溶液中形成multiplemer與其他小分子的complex，最后分子量與蛋白和肽的分子量之和類似。 MALDI可以直接檢測到single charged的complex 峰。 ESI的化看機(jī)器的檢測范圍。如果檢測范圍小于4000,那么直接檢測到的是multiple charged 的分子量，最后通過人工或機(jī)器deconvolution推到出single charged 的分子離子峰。 然后使用LC-MS 分離檢測，如果LC-MS能夠分離檢測到，那么就可以做NMR。對特殊的hydrogen bond進(jìn)行表征。 最后，還可以培養(yǎng)結(jié)合后蛋白和肽的共晶，使用X-ray檢測。 總結(jié)，查相關(guān)類似文獻(xiàn)。 |
3樓2012-07-11 21:44:41
llp159848
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4樓2012-07-12 10:12:21
lzjessie_wlf
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5樓2012-07-12 22:47:54
lzjessie_wlf
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7樓2012-07-12 22:58:39
llp159848
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8樓2012-07-13 09:34:11
lzjessie_wlf
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9樓2012-07-13 20:41:10
lzjessie_wlf
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llp15984810樓2012-07-14 01:56:25
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