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Purification: Difference between revisions
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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 | ||
==Purifying untagged proteins== | |||
A typical strategy for purifying untagged proteins is IEX-HIC-GEC. These three steps are sufficient to produce homogeneous protein from nearly any overexpression system that can produce recombinant protein at >2% of total cellular protein. Variant proteins can typically be purified using a protocol identical to the wild-type, but occasionally single amino acid mutations can produce wildly different chromatographic behavior. | A typical strategy for purifying untagged proteins is IEX-HIC-GEC, in that order. These three steps are sufficient to produce homogeneous protein from nearly any overexpression system that can produce recombinant protein at >2% of total cellular protein. Variant proteins can typically be purified using a protocol identical to the wild-type, but occasionally single amino acid mutations can produce wildly different chromatographic behavior. | ||
==Notes== | ==Notes== | ||
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