FIG. 3.

Purification of endogenous Jhd1 from yeast whole-cell extract. (A) Schematic representation of steps used to purify Jhd1. The numbers represent the salt concentration (in mM) at which Jhd1 eluted from different columns. (B) Silver staining of a polyacrylamide-SDS gel (top panel) and Western blot analysis (bottom panel) of the fractions derived from gel filtration with a Superdex 200 column. Fraction numbers and elution profiles of the protein markers are indicated on the top of the gel. Jhd1 is indicated by an asterisk. (C) Silver staining of an SDS gel (top panel) and Western blot analysis (bottom panel) of the fractions derived from a heparin column after gel filtration. Fraction numbers are indicated on the top of the gel, and Jhd1 is indicated by an asterisk. The # indicates a protein of about 100 kDa that cofractionated with Jhd1 on the heparin column but did not coelute with Jhd1 on the gel filtration column. (D) Hydrodynamic properties of Jhd1. Recombinant Jhd1 was fractionated and analyzed over a 5 to 20% sucrose gradient together with standard molecular weight markers. The sedimentation coefficient of Jhd1 (∼21.37S) was calculated from this sucrose gradient. The Stoke's radius (∼5.16 nm) of Jhd1 was calculated from size-exclusion chromatography. The molecular mass (453.8 kDa) and frictional ratio (1.02) of Jhd1 were calculated using formulas derived by Siegel and Monty (41).