Ni-NTA Cartridge

Catalog No. ABIN4368218

Product Details

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 Details

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

Handling

Storage: 4 °C