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Methods of Protein Iodination
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Horseradish peroxidase catalyzes the oxidation of I- in a pH-dependent manner, optimal rates being observed at low pH (2.7 to 3.6).55,99 With less than stoichiometric quantities of peroxide, an equilibrium mixture of I-, I2, and is obtained.55 Ljunggren100 found no evidence for the enzyme to catalyze the iodination of free tyrosine at pH 7.4, but Pommier et al.101 obtained a good yield of iodotyrosines when working at pH 4.8. Bayse and Morrison55 compared the iodotyrosine-forming activities of horseradish peroxidase and lactoperoxidase, and found that on an equimolar basis, the activity of the former is about 14% of the latter.
Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization
Published in Platelets, 2023
Yu-Wen Wu, Deng-Yao Lee, Yeh-Lin Lu, Liling Delila, Ouada Nebie, Lassina Barro, Chun Austin Changou, Long-Sheng Lu, Hadi Goubran, Thierry Burnouf
Protein concentrations of PEVs and DOX-PEVs were determined using a Pierce BCA protein assay kit (ThermoFisher Scientific). Ten micrograms of proteins was heated at 70°C for 10 min and loaded onto a Precast SurePAGE 4–12% Bis-Tris protein gel (GenScript, Nanjing, China). Proteins were transferred to polyvinylidene difluoride membranes (Pall, Port Washington, NY, USA), and blocked using 5% bovine serum albumin (BSA; Sigma-Aldrich). Membranes were incubated with antibodies against platelet markers CD41 (1:1000, Clone: EPR4330, Abcam, Cambridge, MA, USA) and CD62P (1:108, Clone: AK-6, Abcam), and EV markers CD9 (1:200, Clone: C-4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD63 (1:200, Clone: MX-49.129.5, Santa Cruz). Horseradish peroxidase-conjugated antibodies (1:2000, GeneTex, Irvine, CA, USA) were used as a secondary antibody. Detection was done by enhanced chemiluminescence (ECL) (GE Healthcare). Bands were analyzed on a BioSpectrum 810 Imaging System (Analytik Jena, Jena, Germany).
Blood Levels of Tumor Necrosis Factor Alpha and Its Type 2 Receptor Are Elevated in Patients with Boston Type I Keratoprosthesis
Published in Current Eye Research, 2019
Eleftherios I. Paschalis, Elise V. Taniguchi, James Chodosh, Louis R. Pasquale, Kathryn Colby, Claes H. Dohlman, Lucy Q. Shen
Plasma levels of soluble proteins TNF-α, TNFR1, TNFR2, and leptin were quantified using ultra-sensitive enzyme-linked immunosorbent assay, an enzymatically amplified “two-step” sandwich-type immunoassay (R&D Systems, Minneapolis, MN, USA). Briefly, standards, controls, and unknown plasma samples were incubated in microtitration wells, which had been coated with recombinant human antibody. After incubation and washing, the wells were treated with another anti-human detection antibody labeled with the enzyme horseradish peroxidase. After a second incubation and washing step, the wells were incubated with the substrate tetramethylbenzidine. An acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 and 620 nm. The absorbance measured is directly proportional to the concentration of human protein present. Τhe assay’s sensitivity was 0.106 pg/mL for TNF-α (R&D Systems, Catalog Number HSTA00D), 0.77 pg/mL for TNFR1 (R&D Systems, Catalog Number DRT100), 0.6 pg/mL for TNFR2 (R&D Systems, Catalog Number DRT200), and 7.8 pg/mL for leptin (R&D Systems, Catalog Number: DLP00). Quality controls were performed and all tests were duplicate.
Discovery of novel sulphonamide hybrids that inhibit LSD1 against bladder cancer cells
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Jia Liu, Xingwang Zhu, Liu Yu, Minghuan Mao
LSD1 inhibitor screening assay kit was purchased to evaluate for the enzyme inhibition activity of all synthetic compounds (Cayman Chemical, Fort Annapolis, MI). The MAO-A/B biochemical kit was purchased to explore the inhibitory activity against MAO-A and MAO-B (Promega, Madison, WI). First, compounds J1∼J7, K1∼K3, and L1∼L8 were diluted with dimethyl sulphoxide to required concentrations (Innochem, Beijing, China). In a 48-well black plate, a buffer solution of compounds and reagent was prepared and incubated at 37 °C. Then, horseradish peroxidase solution and fluorometric substrate solution were added and incubated at 37 °C. The signal of each well was read by a GloMax Explorer (Beijing Yuanpinghao Biotechnology Co., Ltd, Beijing, China).