INDIGO Ni-Cartridge

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Application
Purification (Purif)
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Purpose Specific binding and purification of his-tagged proteins
Brand PureCube 100
Sample Type Cell Culture Supernatant, Cell and Tissue Lysate
Specificity Affinity to His-tagged proteins
Characteristics
  • Pre-packed column containing PureCube 100 INDIGO Ni-Agarose
  • Novel His affinity ligand providing stability in 20 mM EDTA and 20 mM DTT
  • Binding capacity up to 80 mg/mL
  • Suitable for high flow rates on FPLC instruments
  •  pH tolerant from pH 4-9.
  • 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
ProductDetails: Bead Ligand Ni-INDIGO
ProductDetails: Bead Matrix Agarose beads
ProductDetails: 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

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
Supplier Images
SDS-PAGE (SDS) image for INDIGO Ni-Cartridge (ABIN4368224) Fig.1: PureCube 100 INDIGO Ni-Agarose can be re-used multiple times without regenerat...