Editing Cre HIC Purification

Hydrophobic Interaction Chromatography 

Hydrophobic interaction (HIC) chromatography is a powerful and often overlooked method of protein purification. This method separates proteins by surface hydrophobicity. HIC is an especially good followup step to IEX, as it requires high salt concentrations for protein binding. Protein solutions should typically be brought to 1.0 M ammonium sulfate in an appropriate buffer. (This can be easily accomplished by slowly adding solid salt to an IEX eluate or crude lysate.) Most proteins will readily stick to an HIC column under these conditions. Choices of chromatographic medium include, in order of increasing hydrophobicity, butylsepharose, octylsepharose, and phenylsepharose. Butylsepharose is a good first choice, as the more hydrophobic media can be too "sticky" for many protein. Elution of protein is accomplished by lowering the ammonium sulfate concentration. For optimization of elution conditions, run a 1-0 M gradient of ammonium sulfate. The final purification protocol should utilize the minimum ammonium sulfate concentration that allows complete binding of the target protein during the loading phase, and the maximum ammonium sulfate concentration that allows for complete elution of the protein during the elution phase. The column should be stripped of protein after use by passing a low ionic strength buffer with no salt through it. A 1.6 x 10 cm HIC column is sufficient to process crude lysate from and IEX pool derived from 1-4 L of bacterial 

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Sample preparation: Dilution 1:1 (v/v) with start buffer <br>Start buffer: 50 mM NaH2PO4, 1.0 M (NH4)2SO4, pH 7.0 <br>Elution buffer: 50 mM NaH2PO4, pH 7.0<br>Gradient: 0–100% elution buffer in 15 CV<br>Flow: 1.0 ml/min (156 cm/h) at room temperature

most commonly used salts are (NH4)2SO4, Na2SO4, NaCl, KCl and CH3COONH4.

Column: HiTrap Butyl FF, 1 ml

Sample loading: 6 mg in 1 ml start buffer<br>Flow: 0.5 ml/min, 75 cm/h<br>Start buffers: 

a) 0.1 M sodium phosphate, 1.7 M ammonium sulfate, pH 7.0<br>b) 0.1 M sodium phosphate, 1.0 M sodium sulfate, pH 7.0<br>c) 0.1 M sodium phosphate, 3 M NaCl, pH 7.

Elution buffer: 0.1 M sodium phosphate, pH 7.0<br>Gradient: 0%–100% elution buffer in 10 CV



start buffer: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0<br>elution buffer: 50 mM sodium phosphate, pH 7.0

equilibration: 5–10 CV<br>Injection <br>wash 5–10 CV <br>gradient elution 10–20 CV <br>salt-free elution buffer 2–5 CV<br>re-equilibration 5–10 CV

Step elution

equilibration: 5–10 CV<br>Injection <br>wash 5–10 CV <br>Step elution 5 CV <br>salt-free elution buffer 2–5 CV<br>re-equilibration 5–10 CV<br><br>

[[Category:Protocols]]