计算溶液所需的质量、体积或浓度。
无细胞蛋白合成试剂盒 (促溶加强版)
有货
为什么选择此级别
BioReagent,用于蛋白分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-80℃储存,避免反复冻融。特低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
该试剂盒是一款基于大肠杆菌细胞裂解液进行体外蛋白质合成的产品。该产品利用细胞裂解液中的核糖体、翻译因子、酶等活性物质，同时补加能量、核苷酸、氨基酸、无机盐等，在体外重构转录-翻译体系。以DNA或RNA为模板，表达出蛋白质。无细胞蛋白表达是一种不依赖于活菌，能够快速、灵活地大量表达蛋白质，和传统的重组表达体系相比有众多的优势：
1. 蛋白表达快，1~2 h即可表达出目的蛋白，8~24 h即可达到最大量。
2. 蛋白表达量高，最高可达到3 mg/ml以上的蛋白。
3. 反应简单灵活，仅需向反应体系中加入DNA或RNA模板，可使用96孔板或离心管进行反应。
注意：本货号产品含有伴侣蛋白，能够促进蛋白可溶性表达。用户开封后，将无细胞蛋白表达反应组分置于-80 ℃储存。为避免反复冻融，可根据反应用量，将A液和B液分别分装后用液氮 速冻后再置于-80 ℃储存。
适用范围
产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗， 不得用于食品或药品。
操作说明
1. 基因构建
目的基因的构建对于蛋白表达至关重要，推荐使用下图的构建方式。可以将目的基因构建到本试剂盒附带的阳性质粒上 (质粒图谱详见外包装标签扫描二维码内文件)，即如下的构建方式；也兼容pET- 9a、pET-23a 等含有T7启动子，但不含乳糖操纵子 (lac)的PET系列质粒。
阳性质粒的DNA序列示意图如下所示：
注：含有lac乳糖操纵子的质粒 (如pET28a等)会对产量有较大影响，不建议直接使用。
图 2 阳性质粒DNA序列示意图
2. 模板制备
无细胞蛋白合成试剂盒可以使用DNA或mRNA作为模板表达重组蛋白。采用的DNA模板可以是质粒，可以是PCR产物，也可以是使用phi29进行RCA滚环扩增的产物。
(1) 质粒：通过基因公司合成，或亚克隆获得适用于无细胞反应的质粒，并采用柱纯化方式提取质粒；
(2) PCR产物：设计引物，正向引物在T7启动子上游200 bp左右，反向引物在终止密码子下游200 bp左右 (包含 T7 terminator)，对模板进行扩增，获得的DNA线性片段可以不经纯化直接投入到无细胞反应体系中。上下游200 bp碱基的作用是保护DNA线性片段不被内源性外切酶降解。
(3) RCA产物：使用phi29聚合酶、随机六引物进行滚环扩增RCA，获得的DNA产物可直接用于无细胞反应体系中。
(4) PCR和RCA可以和Golden Gate以及Gibson Assembly联用，将极大提高DNA模板制备的速度和通量。
(5) DNA模板使用前需要准确定量；建议使用高质量的质粒提取试剂盒进行质粒提取，避免引入RNase A；基因合成公司返回的质粒需强调使用柱纯化方式，否则不能直接用于无细胞反应。
3. 无细胞蛋白表达
(1) 根据反应体系总量计算所需的A液和B液 (体积比1: 2)，将所用的试剂于冰上添加到反应容器中 (例如2 ml圆底离心管)，并混匀。操作过程需全程佩戴手套、口罩，并采用无酶源的移液器吸头和反应容器，避免引入核酸酶。无细胞反应体系可参照下表进行混合配制：
表 2 反应体系的配制
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(2) 向反应体系中加入模板DNA，推荐DNA模板的添加终浓度为5~10 μg/ml，可对DNA模板添加量进行优化。
(3) 将反应容器放置到普通摇床或恒温混匀仪中，进行无细胞蛋白表达，推荐反应温度为25~30 ℃。降低温度会降低蛋白的合成速率，但会增加蛋白的可溶性。 一般反应8 h左右即可达到最大的蛋白产量，也可以过夜反应16 h。当反应温度降低时，应适当增加反应时间。
(4) 无细胞蛋白表达需要充足氧气，当使用2 ml圆底离心管作为反应容器时，反应体系不超过100 μl。无细胞蛋白反应可以等比例放大，放大的反应体系需要使用更大的反应容器，例如摇瓶等，同时保证摇床的转速 (200 rpm)。
4. 检测
反应结束， 取反应液 (检测全部蛋白)或反应上清 (检测可溶蛋白)1 μl左右，进行SDS-PAGE蛋白凝胶电泳检测目的蛋白的表达。
5. 阳性对照
本试剂盒中有绿色荧光蛋白sf-GFP质粒作为阳性对照，可通过肉眼直接观测反应结果。sf-GFP成功表达后，无细胞反应体系将呈现明显的绿色。 如需对sf-GFP进行精确定量，可使用酶标仪进行检测 (Ex/Em=485/528 nm)。
The kit is a product for in vitro protein synthesis based on Escherichia coli cell lysate. This kit utilizes active components in the cell lysate, including ribosomes, translation factors, and enzymes, supplemented with energy sources, nucleotides, amino acids, inorganic salts, etc., to reconstitute a transcription-translation system in vitro. It expresses target proteins using DNA or RNA as templates. Cell-free protein expression enables rapid, flexible and high-yield protein production independent of living cells, and offers numerous advantages over traditional recombinant expression systems:
1. Fast protein expression: Target protein can be produced within 1-2 hours, and maximum yield is achieved in 8-24 hours.
2. High protein yield: Up to more than 3000 μg/mL protein.
3. Simple and flexible reaction: Only DNA or RNA template needs to be added to the reaction system; reactions can be performed in 96-well plates or centrifuge tubes.
Note: This product contains chaperone proteins, which can promote the soluble expression of proteins. After opening, store all cell-free protein expression components at -80 °C. To avoid repeated freeze-thaw cycles, aliquot Solution A and Solution B separately according to usage amount, snap-freeze in liquid nitrogen, and then store at -80 °C.
Application
This product is limited to scientific research use by professional personnel. It shall not be used for clinical diagnosis or treatment, nor for food or pharmaceutical applications.
Protocol
1. Gene Construction
Gene construct design is critical for efficient protein expression. The construction strategy shown below is recommended.The target gene can be cloned into the positive control plasmid supplied with this kit (plasmid map is available via the QR code on the outer package label). The kit is also compatible with pET-9a, pET-23a, and other pET-series plasmids containing the T7 promoter but without the lac operator.
Schematic diagram of the positive control plasmid DNA sequence is shown below:
Note: Plasmids containing the lac operator (such as pET-28a) may significantly reduce protein yield and are not recommended for direct use.
Figure 2 Schematic diagram of positive control plasmid DNA sequence
2. Template Preparation
The Cell-Free Protein Synthesis Kit supports the use of DNA or mRNA as templates for recombinant protein expression. DNA templates may include plasmids, PCR products, or RCA products amplified by phi29 polymerase.
(1) Plasmid DNA: Obtain a suitable plasmid via gene synthesis or subcloning, and purify using column-based plasmid extraction kits.
(2) PCR product: Design primers with the forward primer approximately 200 bp upstream of the T7 promoter and the reverse primer approximately 200 bp downstream of the stop codon (including the T7 terminator). Amplify the template; the resulting linear DNA fragment can be used directly in the cell-free reaction without purification. The 200 bp flanking sequences protect linear DNA from degradation by endogenous exonucleases.
(3) RCA product: Perform rolling circle amplification (RCA) using phi29 polymerase and random hexamer primers. The amplified DNA product can be used directly in the cell-free reaction.
(4) PCR and RCA can be combined with Golden Gate and Gibson Assembly to greatly accelerate and increase the throughput of DNA template preparation.
(5) DNA templates must be accurately quantified before use. High-quality plasmid extraction kits are recommended to avoid RNase A contamination. Plasmids received from gene synthesis companies must be column-purified; otherwise, they cannot be used directly in cell-free reactions.
3. Cell-Free Protein Expression
(1) Calculate the required volumes of Solution A and Solution B (volume ratio=1:2) based on total reaction volume. Add all reagents to the reaction vessel (e.g., 2 mL round-bottom centrifuge tube) on ice and mix gently.Wear gloves and a mask throughout the procedure, and use nuclease-free pipette tips and reaction vessels to avoid nuclease contamination. The reaction system can be prepared according to the table below:
Table 2 Reaction System Composition
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(2) Add template DNA to the reaction mixture. The recommended final concentration is 5–10 μg/mL; the amount can be further optimized.
(3) Incubate the reaction vessel in a shaking incubator or thermomixer for cell-free protein expression. The recommended reaction temperature is 25-30 °C.Lower temperatures reduce the protein synthesis rate but improve protein solubility. Maximum yield is typically achieved after approximately 8 hours; overnight incubation (16 h) is also applicable. Prolong the reaction time appropriately when using lower temperatures.
(4) Cell-free protein expression requires sufficient oxygen. When using a 2 mL round-bottom centrifuge tube, the reaction volume should not exceed 100 μL.The reaction can be scaled up proportionally using larger vessels (e.g., shake flasks) with a shaking speed of 200 rpm.
4. Detection
After completion of the reaction, take approximately 1 μL of the reaction mixture (for total protein) or supernatant (for soluble protein) and analyze target protein expression by SDS-PAGE.
5. Positive Control
The kit includes a sf-GFP (super-folder GFP) plasmid as a positive control, allowing direct visual observation of results.Upon successful expression of sf-GFP, the cell-free reaction mixture will show a distinct green color.For precise quantification of sf-GFP, detection can be performed using a microplate reader (Ex/Em=485 nm/528 nm).
规格
别名
无细胞蛋白合成试剂盒
英文别名
Cell-Free Protein Synthesis Kit | Cell-Free Protein Synthesis Kit (Solubility Enhanced)
规格或纯度
BioReagent,用于蛋白分析
英文名称
Cell-Free Protein Synthesis Kit Pro
应用
蛋白表达
储存与运输
储存条件
-80℃储存,避免反复冻融
运输条件
特低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
| sku | 物理外观 | 组件 | 20 reactions | 100 reactions | Storage |
| C1522810A | 液体 | 无细胞表达体系溶液A (促溶加强版) | 300 μl | 2×750 μl | -80℃，避免反复冻融 |
| C1522810B | 液体 | 无细胞表达体系溶液B | 600 μl | 3×1 ml | -80℃，避免反复冻融 |
| C1522810C | 液体 | CFPS对照质粒 | 2 μg | 2 μg | -80℃，避免反复冻融 |
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0535924 |
分析证书 | C1522810 |
技术文档和文章
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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