Two-Dimensional (2D) PACE Analysis of Fetal Liver Protein Secretion
The liver of one fetus from each gilt was collected into ice-cold minimal essential medium (MEM) with 0.1 times the normal amount of leucine among other modifications. Up to 200 mg of liver tissue was cultured in 5.0 ml of low-leucine MEM with 50 jiCi [3H] leucine or 24 h at 37°C under an atmosphere of 50% N2:45% 02:5% C02. Medium was separated from tissue by centrifugation (1000 X g for 10 min) and then frozen at -20°C until processed further. Conditioned medium from embryonic and fetal livers was dialyzed against 10 mM Tris, 0.02% sodium azide, pH 8.2 (3 changes of 8 L, each overnight) at 4°C. Incorporation of [3H]leucine into nondialyzable macromolecules was determined by scintillation counting of 100 jxl of di-alysate. buy diabetes drugs
A volume of dialysate equal to 10 mg of cultured liver was lyophilized and subjected to 2D-PAGE, Coo-massie blue staining, and fluorography for examination of proteins synthesized by the tissue in culture. Quantification of nondialyzable radioactivity contained in individual protein spots was used to determine differences between ages and treatment groups. Fluorographs (Fig. 1) were aligned with the corresponding dried gels, and spots corresponding to transferrin (TF; 80/6 [Mr X 10_3/pl]), alpha-fetoprotein (AFP; 80/5.2), retinol-binding protein (RBP; 20/5.5), two unidentified proteins (4 [12.5/7.5] and 5 [12.5/6.5]), and background (B; no staining) were punched from the gels using a sharpened 7-mm inner-diameter cork borer. Gel punches were solubilized by incubation in 500 |u,l 30% H202 at 80°C for 48-72 h, quenched with 500 |xl 50 mM ascorbic acid; then 10 ml scintillation fluid was added, and counts per minute (cpm) were determined by scintillation counting. Because nondialyzable radioactivity was not linear with weight of tissue cultured (data not shown), counts from individual spots were expressed as (cpm – cpm B)/total cpm lyophilized.