Fig. 3.

A, four proteins adhere to FABP1 by anti-FABP1 antibody adsorption chromatography. Anti-FABP1 antibodies were bound to beads (see under “Experimental Procedures”) and a column prepared from the treated beads. The proteins from fractions 3–5 of the Sephacryl S-100 HR column were passed over the column, and the column was washed with buffer and the proteins eluted with glycine buffer (pH 2.5). The eluted samples were rapidly neutralized and the proteins concentrated. A, panel 1, Native Gel. The proteins were separated by native PAGE (30 μg) and identified by SimplyBlue SafeStain. All the proteins migrated at 75 kDa as shown. A, panel 2, SDS-PAGE. The proteins from the adsorption column were separated by SDS-PAGE (30 μg) and identified by SimplyBlue SafeStain. The molecular mass of each band is marked. B, proteins bound to the anti-FABP1 antibody adsorption column were identified as Sec13, Sar1, FABP1, and SVIP. Cytosolic proteins from fractions 3–5 of the Sephacryl S-100 HR column were concentrated and pased over an anti-FABP1 antibody adsorption column. The column was washed, and the adherent proteins were eluted as in A. Sample, proteins (30 μg) eluted from the anti-FABP1 antibody adsorption column (A) were separated by SDS-PAGE, transblotted to a nitrocellulose membrane, and sequentially probed with anti-Sec13, Sar1, FABP1, and SVIP antibodies. The membrane was washed between each antibody useage. The proteins and their molecular mass are shown. Wash, after the proteins were applied to the adsorption column, the column was washed with PBS, and the fractions were collected and concentrated, and the proteins (30 μg) were separated by SDS-PAGE. Sec13, Sar1, FABP1, and SVIP were identified in the wash. The molecular mass of each protein is as shown. Note: the wash contained extraneous proteins in addition to Sec13, FABP1, Sar1, and SVIP. The bands were detected by ECL.