21
edits
Purification: Difference between revisions
Jump to navigation
Jump to search
no edit summary
No edit summary |
No edit summary |
||
| (One intermediate revision by one other user not shown) | |||
| Line 8: | Line 8: | ||
=====Methodology===== | =====Methodology===== | ||
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent binding of amino acids to metal ions. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as | Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent binding of amino acids to metal ions. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as Co<sup>2+</sup> , Ni<sup>2+</sup> , Cu<sup>2+</sup> , and Zn<sup>2+</sup> . Most naturally occurring proteins do not have an affinity for metal ions and recombinant DNA techniques are used to introduce this property into a protein of interest. Typically an N- or C-terminal oligohistidine tag of 6-12 histidine residues in length is introduced into the protein sequence. In its most common form, IMAC involves binding of a His<sub>6</sub>-tagged (or simply "His-tagged") protein to a resin charged with Ni<sup>2+</sup> ions. Specificity and affinity of the His-tagged protein binding can be increased charging the resin with Co<sup>2+</sup>, Cu<sup>2+</sup> , or Zn<sup>2+</sup>. | ||
=====Resin types===== | =====Resin types===== | ||
| Line 54: | Line 54: | ||
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 | ||