Abstract
Four column packings for size-exclusion high-performance liquid chromatography (HPLC) of proteins with particle sizes from 3 to 13 μm were compared, using 0.1% sodium dodecyl sulphate in the solvent. Their suitability for the purification of hydrophobic membrane proteins was studied with Sendai virus proteins as a model. The calibration curves of the two 13-μm column packings were linear up to high molecular weights. In contrast to this, large proteins (> 100–150 kD) were eluted later than expected from the 3- and 6-μm packings. Peak capacities for proteins larger than 20 kD ranged from 4.7 to 5.5. Therefore, purification of complex mixtures of membrane proteins will often require rechromatography by a different mode of HPLC. Non-ionic detergents are suitable for further ion-exchange chromatography. The effect of addition of 0.1% of five non-ionic detergents (three gluco-methylalkanamide detergents, octylglucoside, and decyl-polyethyleneglycol-300) to the solvent was investigated and decyl-polyethyleneglycol-300 was found to be most suitable. Size-exclusion HPLC with this detergent resulted in the separation of micelles of three different sizes, of which the larger two contained exclusively the Sendai virus F protein.
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