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Co-NTA Cartridge

Purif Co-NTA Agarose beads 100 μm
产品编号 ABIN4368220
发货至: 中国
  • 应用范围
    Purification (Purif)
    原理
    Specific binding and purification of his-tagged proteins
    品牌
    HighSpec
    样品类型
    Cell Culture Supernatant, Cell Lysate, Tissue Lysate
    特异性
    Affinity to His-tagged proteins
    产品特性
    • Pre-packed column containing HigSpec Co-NTA Agarose
    • Specificity higher than with Ni-NTA
    • Suitable for high flow rates on FPLC instruments
    • Stable in buffer containing 10 mM DTT and 1 mM EDTA
    • Delivered as 5 x prepacked cartridges containing 1 mL bed volume each.
    组件
    Pre-packed affinity cartridges
    试剂未包括
    • FPLC instrument Lysis
    • Wash
    • Elution Buffers
    • Ice bath
    • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
    • 50 mL centrifuge tube
    • Micropipettor and Micropipetting tips
    •  pH meter
    • End-over-end shaker
    • SDS-PAGE buffers, reagents and equipment
      Optional: Western Blot reagents and equipment
    Bead Ligand
    Co-NTA
    Bead Matrix
    Agarose beads
    Bead Size
    100 μm
  • 应用备注
    Technical features of Cartridges:
    - Bed volume: 1 mL
    - Dimensions (mm): 6.2 X 50
    - Body material: Polypropylene
    - Inlet: 10-32 UNF female thread
    - Outlet:10-32 UNF female thread
    说明

    Sample Volume for an assay: >200 mL E.coli culture volume or corresponding quantity. Protocol can be scaled up easily.

    样本量
    200 mL
    实验时间
    4 - 5 h
    实验流程
    Purification of his-tagged protein on FPLC instruments
    试剂准备

    A Purification under native conditions:

    • Native Lysis buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8
      Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer.
      Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1-5 mM.
    • Native Wash buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 20 mM, pH 8
    • Native Elution buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 250- 500 mM, pH 8

      Additional chemicals required: Lysozyme, Benzonase® nuclease,
      Optional: Protease inhibitor cocktail


    B Purification under denaturing conditions:
    • Denaturing Lysis buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 8.0,
      Optional: Benzonase® nuclease (e.g. Merck Milipore, #707464)
    • Denaturing Wash buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 6.3NaH2PO4 100 mM
    • Denaturing Elution buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 4.5
      Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
      Note: Due to urea dissociation, adjust the pH immediately before use.

    实验流程

    Standard protocols for His affinity purification can be applied. Please also refer to the FPLC instrument manufacturer's instructions.

    结果分析

    Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.

    限制
    仅限研究用
  • 储存条件
    4 °C
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