Anti-TYR monoclonal antibody, clone 2G4

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a non-radioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP. METHOD SUMMARY Kinase activity-tagged western blotting is designed to separate and detect multiple kinases that phosphorylate themselves or a specific substrate. Kinases on the blot are subjected to denaturation with guanidine and then stepwise renaturation, prior to phosphorylation using nonradioactive ATP and detection with phosphospecific antibodies.

Enzymatic browning is one of the essential issues affecting the quality of fresh-cut produce. In this study, we identified a novel, safe, and effective anti-browning agent, 3-mercapto-2-butanol, which can prevent fresh-cut potatoes from browning for 5 d under the concentration of 25 mu L L-1 at 5 degrees C. The inhibitory effect of 3-mercapto-2-butanol was close to sodium bisulfite. 3-Mercapto-2-butanol was further identified as a competitive inhibitor which directly inhibited the activity of polyphenol oxidase (PPO) by enzyme kinetic analysis, and the expression levels of PPO-related genes (POT32 and POT33) were decreased after 3-mercapto-2-butanol treatment. Besides, the concentration of enzymatic browning substrate tyrosine (Tyr) in the potato chips was found significantly higher in 3-mercapto-2-butanol treatment group than in the control group. We speculated that 3-mercapto-2-butanol could reduce the consumption of Tyr by inhibiting the activity of PPO. Our finding not only provided a novel and safe anti-browning agent candidate (3-mercapto-2-butanol) to efficiently solve the browning issue for fresh-cut food products, but also preliminarily revealed its mechanism in browning inhibition.