蛋白质组学-无细胞蛋白表达三折页-笔记精简版
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TNT® T7/SP6 Coupled Reticulocyte Lysate System TNT® T7/SP6 Coupled Wheat Germ Extract System TNT® T7/T3 Coupled Wheat Germ Extract System
生物素标记检 测系统
TranscendTM Colorimetric Non-Radioactive Translation Detection System
产品
E. coli S30 Extract System for Circular DNA
E. coli S30 Extract System for Linear Templates TNT® T7 Insect Cell Extract Protein Expression System TNT® T8 Insect Cell Extract Protein Expression System
Wheat Germ Extract Plus
Wheat Germ Extract Plus TNT® SP6 High-Yield Protein Expression System TNT® SP6 High-Yield Protein Expression System TNT® T3 Coupled Wheat Germ Extract System TNT® SP6 Coupled Wheat Germ Extract System TNT® T7 Coupled Wheat Germ Extract System
选择一个无细胞表达系统
选择一个无细胞表达系统
表1. Promega无细胞表达系统比较表：模板、应用及产率
模板
Promega 无细胞表达 系统
是否需要 Kozak RNA DNA RBS 是否更优
Rabbit Reticulocyte
Lysate or Wheat Germ
Extract Systems
普洛麦格（北京）生物技术有限公司
北京市东城区北三环东路36号环球贸易中心B 座907-909 电话：010-58256268 传真：010-58256160 网址：www.promega.com.cn
操作手册编号 TB092 TB102 TM305 TM305 TM306 TM306 TB219 TM045 TM045 TM045 TM045 TM066 TM066 TM282 TM282 TB165 TB165 TB165 TM232
TNT® SP6 Coupled Reticulocyte Lysate System
TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size
TNT® T7 Coupled Reticulocyte Lysate System
TNT® T7 Coupled Reticulocyte Lysate System, Trial Size
Expression Systems Yes Yes Yes
No
参考文献
1. Arduengo, M., Schenborn, E. and Hurst, R. (2007) The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics. In: Cell-Free Expression Kudlicki, W., Katzen, F. and Bennett, R., eds. Landis Bioscience, Austin, TX.
Yes No
Yes
TNT® Coupled
Transcription/
Translation Systems Yes Yes
Yes
TNT® SP6 High-Yield
Protein Expression
System
Yes Yes
Yes
转录分析 Yes No No
S30 E. coli Protein
TNT® T7 Quick Coupled Transcription/Translation System TNT® T7 Quick Coupled Transcription/Translation System, Trial Size TNT® SP6 Quick Coupled Transcription/Translation System TNT® SP6 Quick Coupled Transcription/Translation System, Trial Size
应用
真核细胞蛋白分 析(如：pulldown,
免疫共沉淀)
原核细胞 蛋白分析
Yes
No
结构分析 No
产率
(带有对照的 50ul标准反应
体系)
RRL 50–200 ng; WGE 30–150 ng; WGE Plus 10–80 μg
L4600 L4601 L4610 L4611 L4950 L4960 L5001
植物——麦胚表达 系统
L4151 Rabbit Reticulocyte Lysate, Untreated
L4330 Rabbit Reticulocyte Lysate/Wheat Germ Extract Combination System
L4380 Wheat Germ Extract
L4540 Flexi® Rabbit Reticulocyte Lysate System
2. Brouette, C., Betz, N. and Kobs, G. (2002) Promega Notes 80, 10–3.
3. Zhao, K. et al. (2006) Promega Notes 94, 31–5
图4. 使用FluoroTectTM GreenLys标记系统检测分批模式下处理的 procaspase-3蛋白。带或不带N端HQ标签的Procaspase-3(pF3K WG (BYDV) Flexi®载体)经分批模式于体外合成，采用FluoroTectTM GreenLys 系统进行标记。使用含有100 U (1 μl) 的caspase-8 或 caspase-3 (BioMol) 的裂解物10μl 将Procaspase-3激活，37℃孵育1小时(图A)。使用1 μl 的10 mM HEPES (pH 7.5)处理细胞，取上清(S)做为假处理组，37℃ 孵育1小时。每个泳道相当于1μl最初反应体系所产生的蛋白量。图A， 通过荧光标记或Western分析，procaspase-3的激活模式如图所示。 泳道S，未经caspase-8处理过的上清；泳道A，经过caspase-8处理 过的上清；"FluoroTectTM labeling"图中的泳道M，荧光分子量Marker (Sigma)；"Western Analysis"图中的泳道M，ProSieve®彩色蛋白Marker (Cambrex)。图B，显示位于D9，D28(用于去除结构域前体)和D175(用 于初始激活)的假设的蛋白切割条带。横条中的数字为蛋白片断的分子 量估计值(kDa)；p17为位于D28和D175之间的蛋白片断；p12为蛋白的 C末端片断。图C，凝胶图片显示，37℃孵育1小时，procaspase-3 被 caspase- 8充分消化(泳道 8A)，而procaspase-3 未被 caspase-3 充分消 化(泳道3A)。图D，如果将图C中的反应体系在4℃再孵育24小时，可观察 到procaspase-3能够被其自身成熟形态的酶(泳道3A)进行有限的自消化。 图C和图D中的泳道M，荧光分子量Marker (Sigma)。
150–350 ng
Yes
No
No
(取决于使用哪个系统)
Yes
No
Yes
10–100 μg
100–300 ng
No
Yes
No
(取决于使用哪个系统)
L5010 L5020 L5030 L5040 L5070 L5080 L5511
TNT® T7/T3 Coupled Reticulocyte Lysate System
TNT® T3 Coupled Reticulocyte Lysate System Rabbit Reticulocyte Lysate, Nuclease Treated
荧光标记检测系统
FluoroTectTM GreenLys in vitro Translation Labeling System
昆虫表达系统
S30 T7 High-Yield Protein Expression System S30 T8 High-Yield Protein Expression System E. coli T7 S30 Extract System for Circular DNA
原核——大肠杆菌 表达系统
订货信息
**需要记住的几类无细胞蛋白表达系统
L1102和L1115 T8 写错了，都是T7
哺乳动物—— 兔网织红细胞 表达系统
目录号 L1020 L1030 L1101 L1102 L1110 L1115 L1130 L1170 L1171 L2080 L2081 L3250 L3251 L3260 L3261 L4120 L4130 L4140
Flexi, Monster Green and TNT are registered trademarks of Promega Corporation. FluoroTect and Transcend are trademarks of Promega Corporation. Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. ProSieve is a registered trademark of Lonza Group, Ltd. Whatman is a registered trademark of Whatman Paper Company, Ltd.
L5540 L5600 L5800 L5900
TNT® T7 Quick for PCR DNA Gold TNT® T7 Express 96 System Gold TNT® SP6 Express 96 System
T7 Sample System
兔网织红细胞表达
系统，适合PCR片
段模板
规格 30 reactions 30 reactions 10 reactions 40 reactions 24 reactions 8 reactions 30 reactions 40 reactions 5 reactions 40 reactions 5 reactions 40 × 50µl reactions 10 × 50µl reactions 40 × 50µl reactions 10 × 50µl reactions 40 reactions 40 reactions 40 reactions 30 × 50µl reactions 12 reactions 5 × 200µl 5 × 200µl 40 reactions 8 reactions 40 reactions 8 reactions 40 reactions 5 × 200µl 40 reactions 40 reactions 40 reactions 40 reactions 40 reactions 30 reactions 30 reactions 175µl 40 reactions 1 × 96 wells 1 × 96 wells 1each
TranscendTM Chemiluminescent Non-Radioactive Translation Detection System
Amino Acid Mixture Minus Methionine and Cysteine
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.
TNT® T7/SP6 Coupled Reticulocyte Lysate System TNT® T7/SP6 Coupled Wheat Germ Extract System TNT® T7/T3 Coupled Wheat Germ Extract System
生物素标记检 测系统
TranscendTM Colorimetric Non-Radioactive Translation Detection System
产品
E. coli S30 Extract System for Circular DNA
E. coli S30 Extract System for Linear Templates TNT® T7 Insect Cell Extract Protein Expression System TNT® T8 Insect Cell Extract Protein Expression System
Wheat Germ Extract Plus
Wheat Germ Extract Plus TNT® SP6 High-Yield Protein Expression System TNT® SP6 High-Yield Protein Expression System TNT® T3 Coupled Wheat Germ Extract System TNT® SP6 Coupled Wheat Germ Extract System TNT® T7 Coupled Wheat Germ Extract System
选择一个无细胞表达系统
选择一个无细胞表达系统
表1. Promega无细胞表达系统比较表：模板、应用及产率
模板
Promega 无细胞表达 系统
是否需要 Kozak RNA DNA RBS 是否更优
Rabbit Reticulocyte
Lysate or Wheat Germ
Extract Systems
普洛麦格（北京）生物技术有限公司
北京市东城区北三环东路36号环球贸易中心B 座907-909 电话：010-58256268 传真：010-58256160 网址：www.promega.com.cn
操作手册编号 TB092 TB102 TM305 TM305 TM306 TM306 TB219 TM045 TM045 TM045 TM045 TM066 TM066 TM282 TM282 TB165 TB165 TB165 TM232
TNT® SP6 Coupled Reticulocyte Lysate System
TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size
TNT® T7 Coupled Reticulocyte Lysate System
TNT® T7 Coupled Reticulocyte Lysate System, Trial Size
Expression Systems Yes Yes Yes
No
参考文献
1. Arduengo, M., Schenborn, E. and Hurst, R. (2007) The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics. In: Cell-Free Expression Kudlicki, W., Katzen, F. and Bennett, R., eds. Landis Bioscience, Austin, TX.
Yes No
Yes
TNT® Coupled
Transcription/
Translation Systems Yes Yes
Yes
TNT® SP6 High-Yield
Protein Expression
System
Yes Yes
Yes
转录分析 Yes No No
S30 E. coli Protein
TNT® T7 Quick Coupled Transcription/Translation System TNT® T7 Quick Coupled Transcription/Translation System, Trial Size TNT® SP6 Quick Coupled Transcription/Translation System TNT® SP6 Quick Coupled Transcription/Translation System, Trial Size
应用
真核细胞蛋白分 析(如：pulldown,
免疫共沉淀)
原核细胞 蛋白分析
Yes
No
结构分析 No
产率
(带有对照的 50ul标准反应
体系)
RRL 50–200 ng; WGE 30–150 ng; WGE Plus 10–80 μg
L4600 L4601 L4610 L4611 L4950 L4960 L5001
植物——麦胚表达 系统
L4151 Rabbit Reticulocyte Lysate, Untreated
L4330 Rabbit Reticulocyte Lysate/Wheat Germ Extract Combination System
L4380 Wheat Germ Extract
L4540 Flexi® Rabbit Reticulocyte Lysate System
2. Brouette, C., Betz, N. and Kobs, G. (2002) Promega Notes 80, 10–3.
3. Zhao, K. et al. (2006) Promega Notes 94, 31–5
图4. 使用FluoroTectTM GreenLys标记系统检测分批模式下处理的 procaspase-3蛋白。带或不带N端HQ标签的Procaspase-3(pF3K WG (BYDV) Flexi®载体)经分批模式于体外合成，采用FluoroTectTM GreenLys 系统进行标记。使用含有100 U (1 μl) 的caspase-8 或 caspase-3 (BioMol) 的裂解物10μl 将Procaspase-3激活，37℃孵育1小时(图A)。使用1 μl 的10 mM HEPES (pH 7.5)处理细胞，取上清(S)做为假处理组，37℃ 孵育1小时。每个泳道相当于1μl最初反应体系所产生的蛋白量。图A， 通过荧光标记或Western分析，procaspase-3的激活模式如图所示。 泳道S，未经caspase-8处理过的上清；泳道A，经过caspase-8处理 过的上清；"FluoroTectTM labeling"图中的泳道M，荧光分子量Marker (Sigma)；"Western Analysis"图中的泳道M，ProSieve®彩色蛋白Marker (Cambrex)。图B，显示位于D9，D28(用于去除结构域前体)和D175(用 于初始激活)的假设的蛋白切割条带。横条中的数字为蛋白片断的分子 量估计值(kDa)；p17为位于D28和D175之间的蛋白片断；p12为蛋白的 C末端片断。图C，凝胶图片显示，37℃孵育1小时，procaspase-3 被 caspase- 8充分消化(泳道 8A)，而procaspase-3 未被 caspase-3 充分消 化(泳道3A)。图D，如果将图C中的反应体系在4℃再孵育24小时，可观察 到procaspase-3能够被其自身成熟形态的酶(泳道3A)进行有限的自消化。 图C和图D中的泳道M，荧光分子量Marker (Sigma)。
150–350 ng
Yes
No
No
(取决于使用哪个系统)
Yes
No
Yes
10–100 μg
100–300 ng
No
Yes
No
(取决于使用哪个系统)
L5010 L5020 L5030 L5040 L5070 L5080 L5511
TNT® T7/T3 Coupled Reticulocyte Lysate System
TNT® T3 Coupled Reticulocyte Lysate System Rabbit Reticulocyte Lysate, Nuclease Treated
荧光标记检测系统
FluoroTectTM GreenLys in vitro Translation Labeling System
昆虫表达系统
S30 T7 High-Yield Protein Expression System S30 T8 High-Yield Protein Expression System E. coli T7 S30 Extract System for Circular DNA
原核——大肠杆菌 表达系统
订货信息
**需要记住的几类无细胞蛋白表达系统
L1102和L1115 T8 写错了，都是T7
哺乳动物—— 兔网织红细胞 表达系统
目录号 L1020 L1030 L1101 L1102 L1110 L1115 L1130 L1170 L1171 L2080 L2081 L3250 L3251 L3260 L3261 L4120 L4130 L4140
Flexi, Monster Green and TNT are registered trademarks of Promega Corporation. FluoroTect and Transcend are trademarks of Promega Corporation. Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. ProSieve is a registered trademark of Lonza Group, Ltd. Whatman is a registered trademark of Whatman Paper Company, Ltd.
L5540 L5600 L5800 L5900
TNT® T7 Quick for PCR DNA Gold TNT® T7 Express 96 System Gold TNT® SP6 Express 96 System
T7 Sample System
兔网织红细胞表达
系统，适合PCR片
段模板
规格 30 reactions 30 reactions 10 reactions 40 reactions 24 reactions 8 reactions 30 reactions 40 reactions 5 reactions 40 reactions 5 reactions 40 × 50µl reactions 10 × 50µl reactions 40 × 50µl reactions 10 × 50µl reactions 40 reactions 40 reactions 40 reactions 30 × 50µl reactions 12 reactions 5 × 200µl 5 × 200µl 40 reactions 8 reactions 40 reactions 8 reactions 40 reactions 5 × 200µl 40 reactions 40 reactions 40 reactions 40 reactions 40 reactions 30 reactions 30 reactions 175µl 40 reactions 1 × 96 wells 1 × 96 wells 1each
TranscendTM Chemiluminescent Non-Radioactive Translation Detection System
Amino Acid Mixture Minus Methionine and Cysteine
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.