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sety_xiang金虫 (正式写手)
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[交流]
请教膜蛋白包涵体表达纯化复性
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大家好！我想请教下膜蛋白表达。 我的膜蛋白有15KD，有2个跨膜螺旋，用pET28a表达出来是包涵体，纯化复性效果一直不好，想请各位帮助提供下相关方法或者试剂 盒，谢谢！！ 另外，我还有一个12KD的蛋白，请问用多大浓度的蛋白胶以及哪个蛋白Maker比较好？ 我在网上查到一个Invitrogen公司的native MembraneMax试剂盒，请问有哪位知道它的价格以及使用过它的相关文献吗? 非常感谢大家百忙之中浏览此帖！ |
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HarveyWang
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海外行者
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小木虫(金币+0.5):给个红包，谢谢回帖交流
小木虫(金币+0.5):给个红包，谢谢回帖交流
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在2007年，Wagner S等人利用三种与GFP融合的膜蛋白(YidC、YedZ和LepI)在大肠杆菌BL21(DE3) pLysS中表达，发现这类异源膜蛋白的表达可导致宿主细胞内形成大量的聚集体，其主要成份是过表达的异源蛋白、分子伴侣(DnaK/J和GroEL/S)、蛋白酶(HslUV和ClpXP)以及许多的周质空间和外膜蛋白的前体等的混合物，从而影响细胞呼吸链和三羧酸循环某些酶的组装。 Wagner S., Baars L., Ytterberg A. J., et al. Consequences of membrane protein overexpression in Escherichia coli [J]. Molecular & Cellular Proteomics, 2007, 6(9):1527-1550. 上面已经说了，膜蛋白包涵体里面会有好多其他的蛋白，要用强烈的手段来去除杂质，达到较高的纯度后，可加入一定的 Tween-20 、Triton等辅助溶解和复性。 许多的试剂盒实际上就加入类似的东西来溶解更多的膜蛋白的。。。哈哈哈。。。商业机密。  另外，我还有一个12KD的蛋白，请问用多大浓度的蛋白胶以及哪个蛋白Maker比较好？ 12KD的蛋白，可使用15%的胶（推荐走小分子蛋白的胶Tris-Tricine？）当然，会很靠下面。走小分子蛋白的胶要改变缓冲物质等的配方，你随便Google下就有啦。 我的这个蛋白就是12KD的，15%的胶。 [ Last edited by HarveyWang on 2009-6-26 at 13:32 ] |
2楼2009-06-26 13:13:47
HarveyWang
捐助贵宾 (知名作家)
海外行者
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小木虫(金币+0.5):给个红包，谢谢回帖交流
小木虫(金币+0.5):给个红包，谢谢回帖交流
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在其他地方看到一些资料，粘过来，供大家参考。 Tricine-SDS-PAGE各种浓度外文原配方及具体操作 第一篇： Tricine/Polyacrylamide Gel Electrophoresis Used for pilin processing analysis but generally useful for resolution of small (15-35 Kdal) proteins of similar size. See Strom, M. S., D. N. Nunn, and S. Lory. 1993. A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl. Acad. Sci., 90:2404-2408. or Strom, M. S., and S. Lory. 1992. Kinetics and sequence specificity of processing of prepilin by PilD, the Type IV leader peptidase of Pseudomonas aeruginosa. J. Bacteriol. 174:7345-7351. Original citation: Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379. Reagents: Anode Buffer (+): 200 mM Tris pH 8.9 (can dilute from 10X stock) Cathode Buffer (-): 100 mM Tris/100 mM Tricine/0.1% SDS (no need to pH, but will be ~8.25) these can be made as 10X stocks and diluted before use Gel Buffer: 3.0 M Tris, pH 8.45/0.3% SDS Note: the pH's given for the anode and gel buffers are essential Stacking acrylamide: 48% acrylamide/1.5% bis-acrylamide Separating acrylamide: 46.5% acrylamide/1.5% bis-acrylamide Sample buffer: (add equal volume to sample), for 20 ml: 5 ml 0.5 M Tris, pH 6.8 4 ml 20% SDS 1 ml 2-mercaptoethanol 4 ml 50% glycerol 0.004 g bromophenol blue 6 ml water Pouring Gels For each minigel (Hoeffer "Mighty Small" or BioRad "Mini Protean-II"--scale up as required): 1. Separating gel: 15% gel 2 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 10% gel 1.22 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 0.78 ml water polymerize with 75 ul 10% APS and 7.5 ul TEMED 2. Stacking gel: 0.25 ml stacking acrylamide 0.75 ml gel buffer 2.0 ml water 20 ul 10% APS 2 ul TEMED Polymerize both the stacking and separating gels at the same time (I used small disposable tubes), and pour stacking gel directly onto the separating gel (pour carefully but quickly--they won't mix but the separating gel polymerizes within 2-3 minutes). Make each separating gel mixture separately and add TEMED and APS right before pouring. Multiple stacking gel mixtures can be made in the same tube, but you have around 10 minutes before these start to polymerize too. Sample loading and electrophoresis: For the minigels, 5-10 ul per well gives best results. Layer the samples in the well very carefully, and be sure to flush out any unpolymerized acrylamide before loading. Electrophorese at 25-35 mA (constant current) per gel in minigel setup. Foam will appear on the top (cathode), and additional cathode buffer may have to be added during the run. |
3楼2009-06-26 13:47:43
HarveyWang
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小木虫(金币+0.5):给个红包，谢谢回帖交流
小木虫(金币+0.5):给个红包，谢谢回帖交流
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第二篇 Tricine-SDS-PAGE 1. Assemble each gel sandwich by stacking, in order, the notched aluminum plate, two 0.75-mm spacers, and a glass plate. It is important that the spacers are aligned properly. 2. Fit the gel sandwiches (usually four at a time) tightly in the multiple gel caster. Fill any remaining space in the mold by including additional glass plates. 3. Place the front face plate on the caster, clamp it in place against the silicone gasket, and verify the alignment of the glass plates and spacers. 4. Prepare the separating gel solution as directed below. Do not add TEMED or ammonium persulfate until ready to pour. 5. Fill a 50-ml syringe with the solution and slowly inject it into the caster until the gels are 6 cm high, allowing 1.5 cm for the stacking gel. 6. Overlay each gel with 100 ul 0.1% SDS. Allow gels to polymerize for approximately one hour to overnight. 7. Remove any remaining gel overlay solution by blotting the top of each gel with a piece of Whatman 3mm paper. 8. Prepare the stacking gel solution as described below. Fill a 10-ml syringe with the solution, and inject into each gel sandwich until the top is reached. 9. Carefully place an appropriate comb into each gel, taking care not to trap any bubbles. Allow gel to polymerize for about 1 hour. 10. Remove the front faceplate of the caster. Carefully remove the gels, and separate using a razor blade. o The gels can be stored with the combs in place tightly wrapped in plastic wrap inside a sealable bag at 4°C for 2 to 3 weeks. Keep the gels moist. Do not store gels in the multiple caster. 11. When ready to run the gel, remove the comb and rinse the sample wells with cathode buffer (see below). Place a line indicating the bottom of the each well on the front glass plate, or use a well template. 12. Fill the upper (cathode) and lower (anode) buffer chambers with the appropriate buffer. The upper chamber should be filled to 1 to 2 cm above the notched plate. 13. Using the marks on the glass plate or the well template as a guide, pipette the prepared samples into the wells. 14. Electrophorese the samples at 10 mA constant current per 0.75-mm gel until the dye front reaches the top of the separating gel (45 minutes). Increase current to 20 mA per gel, and continue run until the bottom of the gel is reached (1 hour). REAGENTS USED IN GELS Separating gel monomer (49.5% T 6% C) Mix 93.0 g acrylamide and 6.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days. Stacking gel monomer (49.5% T 3% C) Mix 96.0 g acrylamide and 3.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days. CAUTION: Acrylamide and bisacrylamide are potent neurotoxins and are absorbed through the skin. Wear a mask while weighing the powder. Gloves and a lab coat should be worn when handling the solution. Do not mouth pipette. Anode buffer (0.2 M Tris, pH 8.9) Dissolve 12.11 g Tris base in 200 ml dH2O. Adjust pH to 8.9 with 1 N HCl. Add dH2O to 500 ml total volume. Filter the solution through a 0.45 µm filter and store at 4°C. Cathode buffer (0.1 M Tris, 0.1 M tricine, 0.1% SDS, pH 8.25) Dissolve 6.055 g Tris base and 8.96 g tricine in a total volume of 500 ml dH2O. Filter the solution through a 0.45 µm filter, add 0.5 g electrophoresis-grade SDS, and store at 4°C. It is not necessary to adjust the pH of this buffer, which should be around 8.25. Gel buffer (3.0 M Tris, 0.3% SDS, pH 8.45) Dissolve 181.65 g Tris base and 1.5 g electrophoresis-grade SDS in 300 ml dH2O. Adjust the pH of the solution to 8.45 with concentrated HCl. Bring to final volume of 500 ml with dH2O. Store at 4°C. Stacking gel (4.0% T 3.0% C) In a 50-ml conical tube, mix 0.8 ml of the stacking gel monomer, 2.5 ml of the gel buffer, and 6.7 ml dH2O. Degas under vacuum 10 to 15 minutes. Add 50 µl of 10% ammonium persulfate and 10 µl TEMED. Swirl gently to mix. Use immediately. Produces 10 ml of stacking gel, sufficient for four minigels. Separating gel (16.5% T 6.0% C) In a 50-ml conical tube, mix 10.0 ml of the separating gel monomer, 10.0 ml of the gel buffer, 3.1 ml glycerol, and 6.9 ml dH2O. Degas under vacuum 10 to 15 minutes. Add 100 µl of 10% ammonium persulfate and 20 µl TEMED. Swirl gently to mix. Use immediately. Produces 30 ml of separating gel, sufficient for four minigels. Protocol adapted from Schägger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379, as modified by Lesse, A. J., et al. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Immunol. Methods 126:109-117. |
4楼2009-06-26 13:53:09
HarveyWang
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经典的外文参考文献： Schagger H, Von Jagow G. Anal Biochem, 1987, 166: 368-379 Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis 纳米盘链接： http://img.namipan.com/downfile/ ... Electrophoresis.pdf [ Last edited by HarveyWang on 2009-6-26 at 14:01 ] |
5楼2009-06-26 13:55:43
sety_xiang
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6楼2009-06-29 09:33:44
sety_xiang
金虫 (正式写手)
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7楼2009-06-29 09:35:07
8楼2009-06-29 13:03:42
HarveyWang
捐助贵宾 (知名作家)
海外行者
- BioEPI: 14
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- 在线: 946小时
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- 注册: 2008-03-19
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- 专业: 微生物生理与生物化学
9楼2009-06-29 16:37:23
