IMPACT™ Kit |NEB酶试剂 New England Biolabs

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产品信息

IMPACT Citations

The IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification strategy that utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag (1). It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step, a native recombinant protein without the use of a protease. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on chitin resin (2–4). Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag (Figures 1,2). The vectors included in this kit allow for the fusion of the target protein at its C-terminus (pTXB1) (3,5) or at its N-terminus (pTYB21) (4,6) to the intein tag.

In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications, including protein semisynthesis and site-specific labeling [3,7, Intein Mediated Protein Ligation (IPL, Appendix I)].

Figure 1: Purification of Maltose Binding Protein (MBP) in a single affinity purification step
IMPACT™ Kit |
Lane 1: uninduced cell extract. Lane 2: induced cell extract showing expressed fusion protein. Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C. Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).
Figure 2: Schematic of the IMPACT System
IMPACT™ Kit |
Figure 3: Intein-mediated Protein Ligation (IPL)
IMPACT™ Kit |
Figure 4: Polylinkers in the vectors pTXB1 and pTYB21 
IMPACT™ Kit |
▼ indicates intein cleavage site.
Figure 5: Flow chart for Protein Expression and Purification using the IMPACT System
IMPACT™ Kit |
Sample collection for analysis by SDS-PAGE is indicated.
产品类别:
IMPACT System,
Bacterial E. coli Protein Expression Products,
Chitin Purification (CBD-tag),

Affinity Purification Products,
Protein Purification Products,
Protein Expression Products,

Protein Labeling Products

应用:
Affinity Purification & Expression Tags,
Protein Purification,
IMPACT Affinity Tag,

Peptide Ligation,
Protein Expression in E. Coli,

Protein Expression

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E6901S     Multi-temperature    
        IMPACT™ Kit E6901-1 -20    
        E. coli ER2566 E4130SVIAL -20 1 x 0.2 ml Not Applicable
        Blue Protein Loading Dye B7703AVIAL -20 1 x 1.5 ml 3 X
        pTXB1 Vector N6707SVIAL -20 1 x 0.05 ml 0.2 mg/ml
        pMXB10 Control Plasmid N6903SVIAL -20 1 x 0.05 ml 0.2 mg/ml
        pTYB21 Vector N6709SVIAL -20 1 x 0.05 ml 200 µg/ml
        Anti-CBD Monoclonal Antibody E8034SVIAL -20 1 x 0.05 ml Not Applicable
        DTT B0788SVIAL -20 1 x 5 ml 1 M
        Chitin Resin S6651S 4    
        Chitin Resin S6651SVIAL 4 1 x 20 ml Not Applicable

  • 优势和特性

    Features

    • Single-column purification without the use of proteases to remove the affinity tag
    • Able to produce target protein without vector–derived amino acids
    • Fusion to either C-terminus or N-terminus of target protein
    • Isolation of proteins with or without an N-terminal methionine residue
    • Ligation and labeling of recombinant proteins
    • T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli

  • 相关产品

    相关产品

    • 虾碱性磷酸酶（rSAP）
    • Gibson 组装® 克隆试剂盒
    • T7 表达 E. coli 感受态细胞（高效级）
    • SHuffle® T7 表达 E. coli 感受态细胞
    • NEB® 10-beta E. coli 感受态细胞（高效级）
    • pTWIN1 载体
    • 快速连接™试剂盒
    • 快速末端平齐化™ 试剂盒
    • 瞬时粘性末端连接酶预混液
    • 平末端/TA 连接酶预混液
    • 电转连接酶®

    单独销售的组分

    • 抗 CBD 单克隆抗体
    • 蓝色蛋白上样染料
    • 几丁质树脂
    • pMXB10 Control Plasmid
    • pTXB1 载体
    • pTYB21 载体

  • 参考文献

    1. Chong, S. et al. (1997). Gene. 192, 277-281.
    2. Chong, S. et al. (1998). Nucl. Acids Res.. 26, 5109-5115.
    3. Evans, T.C. et al. (1998). Protein Sci.. 7, 2256-2264.
    4. Watanabe, T. et al. (1994). J. Bacteriol.. 176, 4465-4472.
    5. Muir, T.W. et al. (1998). Proc. Natl. Acad. Sci. USA. 95, 6705-6710.

操作说明、说明书 & 用法

  • 操作说明

    1. Construction of the Fusion Plasmid (E6901)
    2. Primer Design for Restriction Enzyme Cloning (E6901)
    3. Fusion Constructs (E6901)
    4. Fusion Protein Expression (E6901)
    5. Affinity Purification and On-column Cleavage (E6901)
    6. Simplified Expression and Purification Protocol (E6901)
    7. Preparation of Media and Solutions (E6901)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE6901

  • 应用实例

    • Intein-Mediated Protein Ligation IPL and Labeling with the IMPACT™ Kit

工具 & 资源

  • 选择指南

    • IMPACT™ Vectors and Applications
    • Protein Expression and Purification Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. What systems does NEB offer for protein expression and purification?
    2. What is IMPACT?
    3. What are the advantages of the IMPACT System?
    4. What vectors are included in the IMPACT kit?
    5. If my target protein is sensitive to DTT , which vector(s) should I use?
    6. What is Intein-mediated Protein Ligation (IPL)?
    7. What has IPL been used for?
    8. Where can find all IMPACT FAQs?
    9. What gravity column do you recommend?