Hydrophobic interaction chromatography is used to separate proteins with difference in hydrophobicity. This technique is ideally used as intermediate step in a purification process or to capture specific protein. The separation is based on the reversible interaction between the protein and the hydrophobic surface of a chromatographic medium. This reversible interaction is enhanced by using high ionic strength buffer. This makes hydrophobic interaction chromatography an ideal technique to use after precipitation of proteins with ammonium sulphate.
Protein samples in high ionic strength solution bind as they are loaded onto a chromatographic column. Physiological conditions are then altered in such a way that the bound substances or proteins are differentially eluted. These physiological changes are made gradually or with a continuous decreasing salt concentration. Protein of interest is concentrated during binding and then collected them in a purified and concentrated form.
Choice of Hydrophobic Ligand:
Extreme elution conditions may be required to separate very hydrophobic proteins that are tightly bound to very hydrophobic ligands. To avoid such problems hydrophobic media are screened using HiTrap HIC (Hydrophobic interaction chromatography) Test Kit or RESOURCE HIC Test Kit. Depending upon the intensity of the nature of hydrophobicity of the sample proteins media is selected.
Sample Volume and Capacity:
Hydrophobic interaction chromatography technique is independent of sample volume, provided the suitable conditions are selected to bind strongly protein of interest. The total amount of loaded protein and protein that bind to the column should not exceed the total binding capacity of the chromatographic column. For example, during gradient elution, it is advised to use approximately one fifth of the total binding capacity of the chromatographic column.
In hydrophobic interaction chromatography the characteristics of the chromatographic matrix and also the hydrophobic ligand are considered during the selection of the medium. These characteristics are considered along with the other parameters such as sample solubility, scale of purification, required resolution along with the availability of the medium.
Sample preparation method is very important as correct sample preparation will lead to good resolution and also extends the life of the chromatographic column. To ensure efficient binding of the protein during application of the sample, it is advised that the sample should be at the same pH as that of the starting buffer and in high ionic strength solution. For example 1.5M ammonium sulphate or 4M sodium chloride solution. Also care should be taken so that the sample prepared must be free of particulate matter.
It is advised to use small pre-packed columns for the purpose of media scouting and also for method optimization. Pre-packed columns such as HiTrap HIC (hydrophobic interaction column) Test Kit and also RESOURCE HIC Test Kit are ideal for this purpose. Using pre-packed columns will ensure reproducible results along with high performance.
1. First hydrophobic behaviour of the required protein and also its binding conditions must be studied thoroughly and also carefully. Pre-packed columns such as HiTrap HICText Kit or a RESOURCE HICTest Kit are used to select the appropriate medium which gives optimum binding and elution over the required range of the salt (ammonium sulphate) concentration. For the separation of proteins with unknown hydrophobic properties are separated using 0-100% B , that is 0% B is equals to 1M ammonium sulphate.
2. Gradient are selected based on the required resolution
3. Flow rate that maintains resolution and minimises separation time are selected. It is advised to check recommended flow rates for the selected specific medium.
4. For large scale protein purification and also to reduce separation time along with buffer consumption, it is advised to transfer to a step elution immediately after method optimization. In most of the times it is possible to increase sample loading when using step elusion method, this is an additional benefit for large scale purification of required proteins.
If the protein sample adsorb strongly to a gel then physiological conditions which cause conformational changes, such as pH, temperature, chaotropic ions or organic solvents are altered. Conformational changes caused by these agents are very specific to type of the protein.
Cleaning, sanitisation and sterilisation
Cleaning, sanitisation and also sterilization procedures vary depending upon the type of the protein sample and also the medium. Usually these guidelines are supplied with the medium or with pre-packed columns.
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