• 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 5 x prepacked cartridges containing 1?mL bed volume each.

• 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

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

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

• 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.

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

Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.