Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: Separation of proteins with minor charge differences
✍ Scribed by Keith A. Knisley; Dr. L. Scott Rodkey
- Publisher
- John Wiley and Sons
- Year
- 1990
- Tongue
- English
- Weight
- 559 KB
- Volume
- 11
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: Separation of proteins with minor charge differences
Continuous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas C F E system has been used for both ground-based and microgravity separations. The ground-based McDonnell Douglas C F E and RIEF were compared for the ability to resolve mixtures of proteins with known charge differences. Mixtures of 1) cytochrome c, myoglobin, and ovalbumin or 2) beta-lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of C F E and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both C F E and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie-stained analytical I E F gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta-lactoglobulin by C F E was not complete. Ovalbumin was free of beta-lactoglobulin but beta-lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over C F E for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation ofnoncovalent protein:protein complexes which occurs during RIEF separations.