细胞内复性检测
Summary
在这个协议的方法来衡量热休克后细胞内蛋白质复性的描述。这种方法可以用来研究如分子伴侣和他们的共同因素或化合物能够影响他们的活动foldases。萤火虫荧光素酶的活动是作为记者来衡量伴侣复性活动。
Abstract
本协议描述了一个方法来衡量，在基于单元的系统和抑制/刺激活性的化合物可能产生的影响酶的活性分子伴侣。分子伴侣是参与调节蛋白质折叠1的蛋白质，有促进细胞存活时强调侮辱，如热休克2，营养饥饿和接触化学品 /毒药3的关键作用。出于这个原因的伴侣被发现在参与活动，如肿瘤的发展，癌细胞4 chemioresistance以及神经退行性疾病5。能够抑制或刺激这些酶的活性小分子的设计，因此，用于癌症治疗和神经退行性疾病的研究最多的战略之一。这里所描述的实验提供的可能性来衡量一个特定的分子伴侣复性的活动，并研究对比赛的影响ounds就其活动。这种方法调查的分子伴侣基因转染的萤火虫荧光素酶基因的表达载体。它已经描述，可以通过10,11分子伴侣复性变性萤火虫荧光素酶。作为转染控制正常化，为海肾荧光素酶基因的载体转染。在本协议中所述的所有转染基因的X极端 11（罗氏）在HEK – 293细胞。在第一步中，蛋白质的合成，抑制细胞治疗与放线菌酮。此后蛋白展开是在45 ° C诱导热休克30分钟。恢复后，在37 ° C，蛋白质重新折叠成其活性构象和萤火虫荧光素酶的活性是用作读出：会产生更多的光，更多的蛋白质将重新获得原来的构象。非热震惊细胞作为参考（100％的复性荧光素酶）。
Protocol
Discussion
在这项工作中来衡量细胞内分子伴侣复性活动的协议。整个实验可在3到4天，在图概述所示。 1。
萤火虫和海肾荧光素酶产生的光信号的鲁棒性和线性代表一个坚实的基础协议的重现性。
检测的关键步骤是一个有效率的转染试剂的选择，以确保分子伴侣和萤火虫荧光素酶的过度表达。报告基因可交付也与其他方法，如腺病毒?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
达尼洛Maddalo是作为年轻的调查组，从卡尔斯鲁厄理工学院（YIG）的研究奖学金获得者。
Materials
For this experiment a Luminometer form Perkin Elmer ‘1420 Luminescence Counter’ Vector Light′ was used
Programs for the Luminometer:
Renilla: Pump 1, 100 μl injection
Reaction Buffer: Pump1, 70 μl injection
Luciferin: Pump2, 30μl injection
For the buffers prepare these stock solutions in double distilled water:
- – 1M MgSO4 : 246,48g MgSO4*7H2O in 1 liter
- – 0,5M EGTA: 190,18g EGTA in 1 liter
- – 1M KH2PO4: 136,09g KH2PO4 in 1 liter
- – 1M K2HPO4: 228,23g K2HPO4*3H2O in 1 liter
- – 5M NaCl: 292,2g NaCl in 1 liter
- – 0,5M EDTA: 186,12g EDTA*2H2O in 1 liter
GLY-GLY-buffer:
- – 25mM Glycylglycin
- – 15mM MgSO4
- – 4mM EGTA
For 1 liter use 3,3g of Glycylglycin, 15ml of a 1M MgSO4 solution and 8ml of a 0,5M EGTA solution. Solve in water, adjust the pH to 7,8 and bring to a total volume of 1 liter.
Reaction buffer for Renilla/Coelenterazine buffer:
- – 13,4mM KH2PO4
- – 86,6mM KH2PO4
- – 0,5M NaCl
- – 1mM EDTA
For 1 liter use 13,4ml of a 1M KH2PO4 solution, 86,6ml of a 1M K2HPO4 solution, 100ml of a 5M NaCl solution and 2ml of a 0,5M EDTA solution.
| Company | Catalog/Product Number: | |
| DMEM (Dulbecco’s Modified Eagle Medium 1X) | GIBCO | 41966029 |
| Fetal Bovine Serum Gold | PAA | A15-151 |
| X-treme Gene 9 DNA Transfection Reagent |
Roche | 06365809001 |
| PBS Dulbecco’s Phosphate Buffered Saline 1X |
GIBCO | 14190-094 |
| MOPS 3-(N-Morpholino)Propanesulfonic acid |
SIGMA-ALDRICH | M1254 |
| Cycloheximide | SIGMA-ALDRICH | C7698 |
| Passive Lysis Buffer 5X | Promega | E194A |
Glycylglycin |
SIGMA-ALDRICH Roth Roth |
G7278 3054.2 P027.1 |
KH2PO4 |
Roth Roth Roth Roth |
3904.1 6878.2 3957.1 8043.1 |
| Luciferin Firefly | Biosynth | L8200 |
| Coelenterazine | Biosynth | C7000 |
| DTT (Dithiothreitol) | Roth | 6908.2 |
| ATP (Adenosine Triphosphate) | Roche | 10519987001 |
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