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Ni-NTA Cartridge Bead

Purif Ni-NTA Agarose beads 100 μm
Catalog No. ABIN4368218
$348.78
Plus shipping costs $45.00
5 mL ABIN4368218
5 mL ABIN4368218
local_shipping Shipping to: United States
Delivery in 7 to 8 Business Days
  • Application
    Purification (Purif)
    Purpose
    Specific binding and purification of his-tagged proteins
    Brand
    PureCube 100
    Sample Type
    Cell Culture Supernatant, Cell Lysate, Tissue Lysate
    Specificity
    Affinity to His-tagged proteins
    Characteristics
    • Pre-packed column containing PureCube 100 Ni-NTA Agarose
    • High binding capacity up to 80 mg/mL
    • Suitable for high flow rates on FPLC instruments
    • Stable in buffer containing 10 mM DTT and 1 mM EDTA
    • Delivered as 1 x prepacked cartridges containing 5 mL bed volume.
    Components
    Pre-packed affinity cartridges
    Material not included
    • 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
    Ni-NTA
    Bead Matrix
    Agarose beads
    Bead Size
    100 μm
  • Application Notes
    Technical features of Cartridges:
    - Bed volume: 5 mL
    - Dimensions (mm): 11 X 80
    - Body material: Acrylate
    - Inlet: 10-31 UNF female thread
    - Outlet:10-32 UNF female thread
    Comment

    KD of NTA to 6xHis-tag: ca 10 μM

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

    Sample Volume
    200 mL
    Assay Time
    4 - 5 h
    Protocol
    Purification of his-tagged protein on FPLC instruments
    Reagent Preparation

    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.

    Assay Procedure

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

    Calculation of Results

    Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.

    Restrictions
    For Research Use only
  • Storage
    4 °C