无细胞蛋白质合成试剂盒 Transdirect insect cell

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来自昆虫培养细胞的无细胞蛋白质合成试剂盒无细胞蛋白质合成试剂盒                              Transdirect insect cell

Transdirect insect cell

 


  Sf21 昆虫培养细胞在杆状病毒表达系中有不错的成绩,Transdirect insect cell 以其提取液为底物,再活用从动物冲而来的合成合成系,这个系统带有由兔子网状红血球而来的合成系统,克服了合成量少的缺点,是无细胞蛋白质合成试剂盒。

 

 

特点


● 以杆状病毒表达系的 Sf21 细胞为底物

● 编码杆状病毒多角体蛋白基因 5UTR 的高效载体

● 是迄今为止唯一的从动物系的兔子网状红血球而来的试剂盒,蛋白质合成量更高,是普通试剂盒的20倍

无细胞蛋白质合成试剂盒                              Transdirect insect cell

试剂盒内容


● Insect cell Extract(黄色)×5支

● Reaction Buffer(蓝色)×1支

● 4 mM Methionine(红色)×1支

● 0.5 μg/μL Control DNA(白色)×1支

● 0.5 μg/μL Ptd1 Vector(绿色)×1支

● 使用说明书

产品编号 产品名称 产品规格 产品等级
634-07601 Transdirect insect cell
无细胞蛋白质合成试剂盒
40次

Bst DNA Polymerase 链置换酶

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Bst DNA PolymeraseBst  DNA Polymerase                              链置换酶

链置换酶


◆产品说明


  本产品具有5’→ 3’DNA聚合酶活性与链置换活性,是一款自动从模板双链DNA的氢键解离,然后后合成新DNA链的酶。由于其特性,耐热性链置换DNA聚合酶无需DNA的双链解离,即可在恒定温度下合成DNA,且合成不受DNA二级结构的抑制。



特点


●  具有5’→ 3’DNA聚合酶活性与链置换活性

●  可在恒定温度下合成DNA

●  适用于合成GC含量高的DNA链

●  合成不受DNA二级结构抑制


应用


●  利用链置换活性的应用(等温基因扩增法等)



 Bst DNA Polymerase


 理想反应温度

 60°C~65°C

 失活时间*1

 80°C、5 min

*1 将酶原液直接热变性时的失活温度



 来源

 Geobacillus stearothermophilus

 活性

 8 units/μL

 单位定义

 1 unit指以小牛胸腺DNA为引物/模板,在65°C,30 min的条件下将10 nmol的dNTP掺入酸不溶性沉淀物的

 酶量。

 储存液成分

 10 mmol/L Tris-HCl(pH 7.5), 1 mmol/L DTT, 0.1% Tween 20, 0.1 mmol/L EDTA, 50% Glycerol

 酶反应条件

 随酶附带10×的酶反应专用缓冲液(0.5 mL×1)。如反应总体积为50 μL,添加5 μL的酶反应专用缓冲液



◆产品组成


Bst DNA Polymerase(1,600 units)


产品组分

容量

保存

Bst DNA Polymerase(8 units/μL)

200 μL × 1

-20°C

10 × Bst Reaction Buffer(80 mmol/L Mg2+

500 μL × 1

-20°C



◆应用实例


实验案例1  LAMP法中的运用


LAMP法 反应条件


Bst  DNA Polymerase                              链置换酶

  3.0% Agarose 21

  TAE凝胶电泳

  EtBr染色

 

  M:Gene Ladder Wide 1

  1:A 公司Bst DNA Polymerase

  2:本公司Bst DNA Polymerase

 Candidatus Liberibacter asiaticus DNA

1 × 10copies

 FIP*

40 pmol

 BIP*

40 pmol

 F3 Primer*

5 pmol

 B3 Primer*

5 pmol

 Loop Primer F*

20 pmol

 Loop Primer B*

20 pmol

 dNTPs Mixture

1.4 mM each

 10 × Bst Reaction Buffer

2.5 μL

 Bst DNA Polymerase

8 units

 Total

25 μL

Bst  DNA Polymerase                              链置换酶

反应


 65°C

 60 min


*引物序列


FIP: 5'-GCATGCCGAGGATCAATGCCTTGCTTAAAGAGCGTGCTACG-3'

BIP: 5'-TATGCCTAATGGCACGGGGGTAAGCTTCATCCGCCTTCGA-3'

F3 Primer: 5'-TGGGTTAAGTGATGCTGTGG-3'

B3 Primer: 5'-CAACAATATCAGCCCCTGCT-3'

Loop Primer F: 5'-TCTCAACTGTTTCATCAAACCTAGC-3'

Loop Primer B: 5'- CGTGGCGGTTTTTGCTACA-3'



实验案例2  RCA法评估Bst DNA Polymerase的耐热性与理想反应温度


Bst  DNA Polymerase                              链置换酶


RCA法 反应溶液组成


 M13mp18 single strand DNA

40 ng

 Universal primer

50 mM

 dNTPs Mixture

1.4 mM each

 Tris-HCl (pH 8.8 at 25°C)

20 mM

 KCl

10 mM

 (NH4)2SO4

10 mM

 MgSO4

8 mM

 Tween 20

0.10%

 Betaine

0.8 M

 Bst DNA Polymerase

4 units

 Total

20 μL

Bst  DNA Polymerase                              链置换酶

反应条件


 各个温度下

 30 min


结果


Bst  DNA Polymerase                              链置换酶


条带


M: Gene Ladder Wide 1 (产品编号313-06961)

D: 模板 DNA (M13mp8 single strand, 产品编号319-00841)

 

引物序列(Universal Primer)


5'-GTTTTCCCAGTCACGACGTTGTA-3'

 

电泳条件


0.7% Agarose S / TAE凝胶电泳

EtBr染色

使用不含SDS的Loading Buffer

 

备注


  本实验为比较两种Bst DNA Polymerase,使用相同的反应液组成进行反应。在RCA法中使用各种酶配套的专用缓冲液时,须进行预实验探究反应体系。


 

◆相关信息


备注


●  本产品为实验研究用试剂。不可作为医药品使用。

●  检测Candidatus Liberibacter asiaticus的LAMP引物组合是国立研究开发法人 农业、食品产业技术综合研究机构 九州冲绳农业研究中心灵活运用先进技术,在农林水产研究高度化事业“抑制顽固性疾病柑橘绿化病传播的技术开发”中开发的产品。

 

License


●  LAMP(Loop-mediated Isothermal Amplification)法由荣研化学株式会社持有专利权。

 


◆产品列表


产品编号

产品名称

包装

311-07481

Bst DNA Polymerase

1600 units



◆相关产品


产品编号

产品名称

包装

319-07281

Csa DNA Polymerase

1600 units

319-07301

96-7 DNA Polymerase

1600 units

312-07271

dNTPs Mixture(25 mM each)

400 μL

※ 本页面产品仅供研究用,研究以外不可使用。

产品编号 产品名称 产品规格 产品等级

肽核酸(定制合成)

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肽核酸(定制合成)

  PNA是核苷酸通过酰胺键连接形成的肽链,能与DNA互补结合,具有特异的序列。PNA/DNA双链形成的双螺旋结构比DNA/DNA 双链更加稳定。因为难以被生物体内存在的降解酶识别,具有耐降解性。能在大范围的PH值中稳定存在。


  虽然有以上的几种特点,但在实际应用中,通过一般的自动合成器聚合而成的纯度并不理想。

肽核酸(定制合成)


◆以往的PNA合成中存在的问题

1、组件价格昂贵

2、PNA 聚酰胺构架形成酰胺键的时候,保护基容易发生位阻。(合成困难的位点很多)

3、所使用单体的纯度对目标PNA的生物活性影响大

  HiPep 研究所用全新开发合成法提供高纯度 PNA 产品。

◆特点

• 能提供高纯度的单体原料(97% 以上)

• 通过使用新型独自开发的 PetiSyzer®(小型多种类项目合成器),能合成高纯度的 oligomer。

• 从结果来看,能提供高活性的肽核酸,解决以往核酸肽 PNA 的纯度问题

合成氨基酸的数量范围

标准的合成范围为:10~15 base

16 base 以上是由于正常产量偏低,属特殊订单。请与我们联系。

品质保证的数据

反相 HPLC 分析数据和质谱分析数据标准附件

合成样本的精制纯度

精制品(保证 80~90% 的纯度)

标准的合成量

最低保证量 150 μg

除上述以外为特殊订货,请与我们联系。

供货形式/储存条件

冷干品

◆应用设计例子

肽核酸(定制合成)

PNA 生物结合物(Modular Type)可以通过导入各种细胞渗透性肽和间隔序列来达成目的。通过结合细胞渗透性肽能改善细胞向内运输和核易位。而且,通过引入酶切序列释放药物,能应用于 DDS 和成像。

产品编号 产品名称 产品规格 产品等级

重组无细胞蛋白合成系统 PUREfrex® 2.0

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重组合无细胞蛋白合成系统重组无细胞蛋白合成系统                              PUREfrex® 2.0

PUREfrex® 2.0

 

◆简介


  PUREfrex® 试剂盒是在东京大学的Takuya Ueda教授所发明的PUREsystem技术基础上,新开发的一款重组合无细胞蛋白合成试剂盒。

  反应系统由蛋白质、核糖体、氨基酸和NTPs组成[1,2],其中蛋白行使转录、翻译和能量供应的功能。蛋白与核糖体为分别单独高度纯化后,再重新组合成蛋白合成系统,而非直接从大肠杆菌S30中提取。当合成蛋白时,仅需将编码目的蛋白的模板DNA或mRNA添加到反应混合液中并孵育数小时,即可完成反应。本系统的突出特色是在体外以转录相关因子重新组合一套表达系统,并可根据需要来调整反应混合物的成分,而不必担心高背景会影响下游的应用。进行蛋白表达仅需将编码目标蛋白的模板DNA或mRNA加入到反应体系中,然后孵育2-4小时即可完成反应。PUREfrex® 试剂盒的所有蛋白组分均不带标签,因此目的蛋白可融合任意标签进行纯化和检测。


重组无细胞蛋白合成系统                              PUREfrex® 2.0

 

>>>无细胞表达的优势<<<


● 无需制备克隆

● 无需考虑培养条件

● 无需考虑表达所需的诱导条件

● 来源于宿主的污染少

 


◆PUREfrex® 系列


● PUREfrex® 1.0 第一代产品    

● PUREfrex® 2.0 第二代产品,表达量更高,污染水平更低;

● RNA酶与β-半乳糖苷酶污染大大降低;

● 每1 µL反应混合物中的脂多糖(LPS)低于0.1 EU。

● PUREfrex® 2.1 更适合二硫键的形成



☆升级至PUREfrex® 2.0


1.合成原核和真核蛋白的结果显示,用PUREfrex® 2.0合成时,各种蛋白的合成量增加。


重组无细胞蛋白合成系统                              PUREfrex® 2.0

2.GFP蛋白合成的结果显示,用PUREfrex® 2.0合成时,可以观察到荧光强度增强了10倍以上(每单位反应产物)。


重组无细胞蛋白合成系统                              PUREfrex® 2.0

3.合成需要形成二硫键(SS键)的大肠杆菌酸性磷酸酶(AppA1)时,在PUREfrex® 2.0基础上,添加了DS supplement的结果显示,存在

3.氧化剂和二硫键异构酶时,活性蛋白合成量增加。


重组无细胞蛋白合成系统                              PUREfrex® 2.0


1:AppA有5个二硫键,是其中一个位点在不连续的半胱氨酸之间存在的二硫键。



4.正确高级结构蛋白的合成结果显示,存在分子伴侣2的情况下用PUREfrex® 2.0,蛋白合成量增加。


重组无细胞蛋白合成系统                              PUREfrex® 2.0


2 DnaK Mix:DnaK / DnaJ / GrpE mixture为配套分子伴侣

 

◆特点


● 可以同时加入多种模板进行反应,以合成Fab(带二硫键)及多聚体等带二级结构的多肽

● 可合成活细胞难以合成的强毒性蛋白

● 可直接使用PCR产物来作为模板DNA

● 单位体积内合成的蛋白量几乎恒定,不随反应体积变化而产生显著差异

● 操作简便,仅需在37℃孵育数小时

● 可以合成带标签的蛋白用于下游纯化和检测

● 产品经优化升级,合成量大大提高

 

◆应用


制备目的蛋白


● 原核蛋白

● 真核蛋白

● 膜蛋白

● 二硫键蛋白

● 含有非天然氨基酸的蛋白质等

 


蛋白基础研究


● 翻译

● 蛋白合成后折叠

 


体外展示技术


● 核糖体展示技术

● mRNA展示技术

 


◆应用实例


利用PUREfrex® 系统合成并一步纯化DHFR-His


重组无细胞蛋白合成系统                              PUREfrex® 2.0


3:模板DNA的构建方法请见"相关资料"栏或点击这里



◆添加剂(用于需要形成二硫键和分子伴侣的蛋白质)


● DS supplement

● 通过添加DS supplement至PUREfrex® 反应液中,为二硫键形成创造理想环境。DS supplement作为创造氧化环境的氧化剂,含有氧化型

● 谷胱甘肽(GSSG)和作为二硫键异构酶的大肠杆菌DsbC。当蛋白需要二硫键才能产生活性形式时,请使用本添加剂。

 

● DnaK Mix

● DnaK Mix是高度纯化后的大肠杆菌来源的DnaK、DnaJ、GrpE蛋白以适当的浓度比例预混后的溶液。在PUREfrex® 反应体系中单独或添加

● DS supplement合成蛋白时同时添加DnaK Mix,可以更易获得难以独自形成高级结构的活性蛋白。

 

● GroE Mix

● GroE Mix是高度纯化后的大肠杆菌来源的GroEL、GroES蛋白以适当的浓度比例预混后的溶液。以PUREfrex® 反应体系合成蛋白时添加

● GroE Mix,可以更易获得难以独自形成高级结构的活性蛋白。

 

◆试剂盒组成


用于250 μL反应

使用前请将试剂盒置于-80°C保存

试剂

体积

成分说明

保存温度

溶液 I (白盖

125 μL

氨基酸,核苷酸,tRNA和酶的底物等

-20°C

溶液 II (黑盖

12.5 μL

蛋白,保存于含30%甘油的缓冲液

-20°C or -80°C(1)

溶液 III (红盖

12.5 μL ×2

核糖体(20 μM)

-80°C(1)

DHFR   DNA (透明盖)(2)

10 μL

对照DNA,含有编码大肠杆菌DHFR基因的PCR产物(20 ng/μL)

-20°C


(1)剩余的溶液应快速在液氮、干冰或乙醇中冻结,并储存于-80℃。如有必要,分装剩余溶液,并尽可能避免反复冻融。

(2)每50 μL反应中加入2.5 μL DHFR DNA。

 

◆产品列表

产品编号

产品名称

规格

备注信息

GFK-PF201-0.25-EX

PUREfrex® 2.0

1 kit

供250 μL反应使用

GFK-PF201-0.25-5-EX

1 kit

供250 μL×5次反应使用

GFK-PF213-0.25-EX

PUREfrex® 2.1

1 kit

供250 μL反应使用

GFK-PF213-0.25-5-EX

1 kit

供250 μL×5次反应使用

GFK-PF003-0.5-EX

DnaK Mix

1 kit

供500 μL反应使用

GFK-PF004-0.5-EX

GroE Mix

1 kit

供500 μL反应使用

GFK-PF005-0.5-EX

DS supplement

1 kit

供500 μL反应使用

 

相关产品的详细信息请点击查看:重组无细胞蛋白合成系统 PUREfrex® 2.0


相关资料

重组无细胞蛋白合成系统                              PUREfrex® 2.0 重组无细胞蛋白合成系统                              PUREfrex® 2.0

PUREfrex™ Technical information

PUREfrex™ Protocol

蛋白质工程相关产品

PUREfrex:重组无细胞蛋白合成试剂盒

RYTS试剂盒:大肠杆菌无细胞蛋白质合成系统

CloverDirect:定点蛋白质功能化tRNA试剂

纯化系统:一步高纯度标记纯化系统

STELLA +“赖氨酸标记试剂盒”

  • PUREfrex : Reconstituted Cell-free Protein Synthesis Kit

  • RYTS Kit : E. coli Cell-free Protein Synthesis System

  • CloverDirect : tRNA Reagents for Site-Directed Protein Functionalization

  • Dock Purification System : One step high purity purification tag purification system

  • STELLA+ " Lysine Labeling Kit "

PUREfrex Q&A

Q: 使用PUREfrex™ 试剂盒是否可用于真核蛋白的合成?

: PUREfrex™ 是由E.coli的核糖体和翻译因子组成的体外重组蛋白合成试剂盒,但也可以合成哺乳动物和植物的蛋白。目标蛋白的合成效率         取决于编码蛋白的核苷酸序列,比如GC含量,稀有密码子的含量。

 

Q: 使用PUREfrex™ 试剂盒可以合成多少蛋白?

: 这个取决于目标蛋白。来自E.coli的二氢叶酸还原酶每毫升反应液可合成150 μg。

 

Q: 是否可以合成大于100 kDa的蛋白?

A: 我们用该试剂盒合成了116 kDa的蛋白。

 

Q: 是否可以推荐PUREfrex™ 的反应条件?

A: 推荐用该试剂盒在37℃反应2~4小时。

 

Q: 是否可以合成和纯化标签蛋白?

A: 可以使用任何标签,PUREfrex™ 试剂盒的所有蛋白成分都没有用于纯化或者检测的标签。比如,合成后可用金属螯合的树脂纯化带有His 

       标签的目标蛋白。

 

Q: 合成蛋白是否经糖基化或者磷酸化修饰?

A: 不。不会发生翻译后修饰,PUREfrex™ 试剂盒只是由翻译因子组成。

 

Q: PUREfrex™ 试剂盒是否含有分子伴侣?

A: 不。PUREfrex™ 试剂盒不含有任何分子伴侣,但你可以添加分子伴侣,比如Hsp70。你可以自己制备。

 

Q: 用PUREfrex™ 试剂盒是否可合成含有二硫键的蛋白?

A: 不行。目标蛋白合成不带有二硫键,因为翻译反应时有还原剂DTT。大多数需要二硫键才有活性的蛋白,会没有活性。

 

Q: PUREfrex™ 是否可合成膜蛋白?

A: 一般情况,合成膜蛋白会形成聚集。为了获得能够插入到脂双层的膜蛋白,需要在合成膜蛋白时添加脂质体到PUREfrex™。

 

Q: 是否可合成带有[35S] 甲硫氨酸或者 [3H] 亮氨酸的蛋白?

A: 添加放射性元素标记的氨基酸可以合成放射性元素标记的蛋白,比如[35S] 甲硫氨酸或者 [3H] 亮氨酸。PUREfrex™ 含有20种天然的氨基

       酸,浓度都在0.5 mM。请优化条件。

 

Q: 除了T7启动子外,是否可用其他启动子?

A: 我们推荐使用T7启动子的模板DNA,因为PUREfrex™ 含有转录的RNA聚合酶。当你使用其他聚合酶,制备的模板DNA要有相应聚合酶的

       合适启动子。

 

Q: 使用DHFR DNA(阳性对照)无法获得DHFR。

A: 该试剂盒由于某些原因失活。为了避免失活,请将该试剂盒存放在适当稳定。可进行分装,避免反复冻融影响试剂盒的使用效果。或者改

        试剂盒被核酸酶污染了。请使用不含核酸酶的水,试剂和材料。

 

Q: 使用试剂盒的DHFR可以得到DHFR。但是不能得到目标蛋白,或者目标蛋白量很低。

A: 1)改试剂盒由于某些原因失活了。为了避免失活,请将该试剂盒存放在适当的温度并且进行分装(避免反复冻融)

A: 2)可以受核酸酶污染。为了避免核酸酶污染,请使用不含核酸酶的水,试剂和材料。

A: 3)制备的DNA模板不准确。需要制备含有T7启动子,核糖体结合位点,起始密码子,终止密码子的DNA模板。

A: 4)转录的二级结构会阻止翻译反应。这种情况,请优化模板的顺序,解决二级结构的问题。

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产品编号 产品名称 产品规格 产品等级
GFK-PF201-0.25-EX PUREfrex® 2.0 1 KIT
GFK-PF201-0.25-5-EX PUREfrex® 2.0 1 KIT
GFK-PF213-0.25-EX PUREfrex® 2.1 1 KIT
GFK-PF213-0.25-5-EX PUREfrex® 2.1 1 KIT
GFK-PF003-0.5-EX DnaK Mix 1 KIT
GFK-PF004-0.5-EX GroE Mix 1 KIT
GFK-PF005-0.5-EX DS supplement 1 KIT