| Abstract: | A high-performance liquid chromatographic procedure for recovering subnanomole amounts of protein from SDS/polyacrylamide gel electroeluates in a form suitable for gas-phase sequence analysis has been developed. By a judicious choice of reversed-phase column packing, proteins can be retained at high concentrations of n-propanol (90-100%) where sodium dodecylsulfate and acrylamide gel-related contaminants are washed through the column. Retained proteins can be recovered from the column in high yield (greater than 90%) by the simultaneous adding of an ion-pairing reagent into the mobile phase and elution with a gradient of decreasing n-propanol concentration (i.e. an 'inverse or negative gradient'). Furthermore, by using a steep gradient (e.g. 50%/min) at a low flow rate (20-200 microliters/min) the proteins can be recovered in less than 100 microliters and can be used for gas-phase sequence analysis without further manipulation. This procedure is independent of sodium dodecylsulfate concentration (up to 1.2% w/v) in sample loading volumes of up to 1.5 ml. Microbore columns (2.1 mm internal diameter) have been employed for recovering small amounts of protein (1-100 micrograms from electroeluates of protein-containing gel spots while conventional columns (4.6 mm internal diameter) were used for isolating larger amounts of protein (greater than 500 micrograms) from electroeluates of preparative gel bands. The general utility of this inverse-gradient high-performance liquid chromatography procedure has been demonstrated by its successful application in recovering a wide variety of proteins from sodium dodecylsulfate gel electroeluates in a form suitable for N-terminal sequence analysis in the 10-500 pmol range. |
