Figure 7
Oxidative modifications to proteins in postoperative PCF. (a) Representative in-gel fluorescence image of individual patient PCF (0 and 4 h) treated with 500 μ M Bodipy-iodoacetamide (Bodipy-IAM) for 30 min to assess protein thiol modifications, where lower fluorescence intensity represents increased oxidized protein thiols. Following alkylation, 5 μg of protein was resolved by 12.5% SDS-PAGE and imaged in-gel using a Typhoon imager. The bottom panel shows coomassie blue stain to confirm equal protein loading. Quantitation of the Bodipy-IAM fluorescence signal for albumin normalized to coomassie blue protein staining; n=7 patients. Data expressed as mean±s.e.m. *P<0.05 compared with 0 h PCF. (b) Carbonylated proteins (100 μg) from 0- and 4-h albumin-depleted PCF were derivatized with biotin hydrazide (1 mM) for 30 min at 37 °C. Reactions were stopped with 1 M Tris-HCl, pH 7.4 and proteins (10 μg) separated using SDS-PAGE. The extent of carbonylation was determined with western blot analysis using Streptavidin. The quantification of lane intensities is shown to the right.;n=4 patient matched sets. Statistical significance was measured using Student’s t-Test. Data expressed as mean±s.e.m. *P<0.05, **P<0.005.
