Hydrophobic interaction chromatography is a well-established method to purify proteins according to their surface charge and hydrophobicity. Chromatography matrices that provide a hydrophobic surface are used for separation. This method does not require any affinity tags to be fused to the protein to be purified. Therefore it is suitable for the purification of both native and recombinant proteins. We offer hydrophobic interaction matrices, with phenyl, octyl, or butyl groups on the surface. For a novel protein, comparing different matrices is strongly recommended, and a HIC screening set with small quantities of each matrix is available. Our PureCube Agaroses are based on BioWorks Workbeads, providing highest binding capacities and reproducible results, especially in FPLC chromatography applications.
PureCube Hydrophobic Interaction Agaroses provide:
- Increased ligand density for high protein yields, especially for smaller proteins
- Choice of different matrices: Phenyl, Octyl, and Butyl Agarose
- Homogeneous base matrix made of 40 µm diameter, crosslinked agarose
- Ideal as second step after purification with Ion Exchange Agaroses
How does hydrophobic interaction (HIC) work for protein purification?
HIC separation is based on the interaction of hydrophobic regions of a protein with chromatography media that contain hydrophobic groups such as butyl, octyl, or phenyl on the surface. This interaction is enhanced by high ionic strength, making HIC an ideal purification step after ammonium sulfate precpitation or after elution from ion exchange media in high salt buffers.
Proteins might precipitate at very high salt concentrations, therefore it is recommended to test in small scale experiments if the protein of interest stays in solution at the salt concentration required for binding.
Bound proteins can be eluted with low-salt buffers. It is highly recommended to use a salt gradient (e.g. ammonium sulfate or sodium chloride) for elution to ensure that the protein is kept in the optimal salt concentration required for stability.
Which matrix for my protein?
In contrast to isoelectric point determination, it is difficult to predict a protein's overall hydrophobicity, as only isolated patches on the protein surface may be hydrophobic but sufficient for interaction with the HIC matrix. Therefore, it is recommended to test different resins for their suitability to bind the protein of interest at concentrations that keep the protein in solution, elute it at salt concentrations suitable for protein stability and activity, while separating it from contaminating proteins. The PureCube HIC Starter set was developed for easy comparison of the three different materials.
Fig. 1: Chemical structures of functional groups used in hydrophobic interaction matrices.
Fig. 2: Purification of two test proteins on PureCube Phenyl Agarose and a leading competitor product. For the larger test protein (BSA, 66 kDa) yields of about 25 mg/mL resin were obtained with both HIC resins. For the smaller test protein (lysozyme, 14 kDa) yields were about 4x higher on the PureCube matrix (PureCube: 45 mg/mL, competitor G: 11 mg/mL). Purification was done in batch, with binding and washing at 1.5 M ammonium sulfate, 100 mM NaH2PO4, pH 7.0, and elution at 100 mM NaH2PO4, pH 7.0. E1, E2: elution fractions 1 and 2.
This list includes features that apply to all HIC agarose products. If a difference exists in a specific feature it is mentioned.
|Usage ||Binding to protein via hydrophobic interactions |
|Specifity ||Hydrophobic residues in proteins |
|Flow rates ||Up to 6 mL/min |
|Filling quantity ||Delivered as a 50 % suspension |
|pH stability ||pH 2-13 |
|Chemical group density ||40 µmol/mL |
|Binding capacity ||BSA 25 mg/mL (10mg /mL for Octyl); Lysozyme 45 mg/mL (20 mg/mL for Octyl). |
|Bead size ||40 μm |
|Bead Ligand ||Phenyl, Octyl or Butyl groups |
|Required equipment || |
- Lysis Buffer
- Wash Buffer
- Elution Buffer
- Refrigerated centrifuge for 50 mL tubes (min10,000 x g) and 2 mL tubes
- Refrigerated superspeed or ultracentrifuge capable of 100.000 x g
- End-over-end rotator
- 2 mL microcentrifuge tubes
- 15 mL polypropylene tube, 50 mL polypropylene tube and 50 mL polycarbonate high speed centrifuge tube (for ultracentrifuge)
- Micropipettor and Micropipetting tips
- Disposable gravity flow columns with capped bottom outlet, 2 mL
- pH meter
- End-over-end shaker
- SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment