Aspartate aminotransferase ast activity assay kit

In order to analyze the specific activity of PfAspAT and PfMDH, we performed activity assays in parasites lysates. For this analysis, cultures of transgenic cell lines pARL‐PfAspAT‐WT, pARL‐PfAspAT‐Y68A/R257A, pARL‐PfMDH‐WT, and pARL‐PfMDH‐V190W, as well as the WT 3D7 culture used as a control, were isolated via saponin lysis.
The specific activity of PfMDH was measured with the Malate Dehydrogenase Assay Kit (Sigma Aldrich). The reaction was carried out at 37°C in a final volume of 150 µl, according to the manufacturer's protocol. The absorbance was monitored at 450 nm.
The specific activity of PfAspAT was measured with the Aspartate Aminotransferase (AST) Activity Assay Kit (Sigma Aldrich). The reaction was carried out at 37°C in a final volume of 100 µl, according to the manufacturer's protocol. The absorbance was also monitored at 450 nm. The amount of total protein in the lysates was quantified by Bradford assay (Bradford, 1976).

To assess indices of tissue injury, blood was collected from mice 24 h following sham or CLP procedure via cardiac puncture using a heparin-coated syringe. Blood sample was centrifuged at 10,000 rpm, and supernatant (plasma) was collected and stored at -80°C. For tissue injury assays, plasma was analyzed using the following kits according to the manufacturer’s protocol: Urea Nitrogen (BUN) Colorimetric Detection Kit (Invitrogen, Carlsbad, CA, USA, cat# EIABUN), Creatine Kinase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA: cat# MAK116), Alanine Aminotransferase (ALT) Activity Assay Kit (Sigma-Aldrich: cat# MAK052), Aspartate Aminotransferase (AST) Activity Assay Kit (Sigma-Aldrich: cat# MAK055), Amylase Assay Kit (Colorimetric) (Abcam, Cambridge, MA, USA: cat# ab102523), and Bilirubin Assay Kit (Direct Colorimetric) (Abcam: cat# ab235627).

At 25 weeks, blood was obtained by cardiac puncture under deep naesthesia using isoflurane. Serum was separated by centrifugation (3000 rpm, 10 min). Enzyme-linked immunosorbent assays for mouse insulin (EZRMI-13K; Millipore, Boston, MA, USA) were undertaken according to the manufacturer’s instructions. ALT activity was measured using an alanine aminotransferase activity assay kit (Sigma, St. Louis, MO, USA). Serum aspartate aminotransferase activity was measured using an aspartate aminotransferase (AST) activity assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions.