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Purification: Difference between revisions
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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 culture. | 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 culture. | ||
===Gel Exclusion Chromatography=== | ===Gel Exclusion Chromatography (also termed Size Exclusion Chromatography or Gel Filtration)=== | ||
Gel exclusion chromatography (GEC) separates proteins by size (volume). GEC is an especially good followup step to IEX or HIC, as it can desalt protein preparations, and is typically used as a "polishing" step near the end of a purification. For maximum resolution, a large column should be used (2.6 x 60 cm is typical) and protein should be concentrated to <4% of the total column volume, and flow rates should be as slow as practical. A typical loading for a 2.6 x 60 cm Superdex 200 column is no more than 2.0 mL, and a typical flow rate is 1 mL/min. It is advisable to include 100 mM NaCl or another salt to suppress non-specific binding of protein to the chromatographic medium | Gel exclusion chromatography (GEC) separates proteins by size (volume). GEC is an especially good followup step to IEX or HIC, as it can desalt protein preparations, and is typically used as a "polishing" step near the end of a purification. For maximum resolution, a large column should be used (2.6 x 60 cm is typical) and protein should be concentrated to <4% of the total column volume, and flow rates should be as slow as practical. A typical loading for a 2.6 x 60 cm Superdex 200 column is no more than 2.0 mL, and a typical flow rate is 1 mL/min. It is advisable to include 100 mM NaCl or another salt to suppress non-specific binding of protein to the chromatographic medium | ||