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Immunology
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
It is noteworthy that these studies were performed by the most advanced modern methods. Thus, the melting temperature of the sdAb and sdAb–alkaline phosphatase fusion proteins was measured by a combination of circular dichroism, differential scanning calorimetry, and fluorescence-based thermal shift assay, while binding kinetics were assessed using surface plasmon resonance.
Cepharanthine hydrochloride induces mitophagy targeting GPR30 in hepatocellular carcinoma (HCC)
Published in Expert Opinion on Therapeutic Targets, 2020
Yao Wang, Gui-Feng Su, Ze-Xiu Huang, Zhen-Guang Wang, Peng-Jun Zhou, Jiang-Lin Fan, Yi-Fei Wang
Based on the biophysical principle of ligand-induced thermal stabilization of target proteins, ITDRF assay was used to estimate the concentration effects of CH on its potential target protein GPR30 [28]. Briefly, phosphate-buffered saline (PBS) diluted cell suspensions were freeze-thawed three times in liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20,000 × g for 20 min at 4°C. The cell lysates were diluted with PBS and divided into five aliquots, with one aliquot (50 μl) treated with DMSO and other aliquots with increasing concentrations of CH. After incubation for 30 min at room temperature, the respective lysates were heated at constant temperature of 50°C for 3 min followed by cooling for 3 min at room temperature. The appropriate temperature was determined in preliminary cellular thermal shift assay (CETSA) experiments (data not shown) [28]. The heated lysates were centrifuged at 20,000 × g for 20 min at 4°C, and the supernatants were then mixed with 5× loading buffer and denatured at 100°C for 5 min. Equal volume of the denatured lysate was subjected to SDS-PAGE followed by Western blotting analysis.
Prolonging the shelf life of Lumbricus terrestris erythrocruorin for use as a novel blood substitute
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Christine Muzzelo, Christopher Neely, Payal Shah, Osheiza Abdulmalik, Jacob Elmer
Melting temperatures (Tm) were measured with a thermal shift assay utilizing SYPRO orange dye, which fluoresces when it binds to hydrophobic residues that are exposed as proteins denature at high temperatures (Lavinder et al. 2009). For this assay, each sample was diluted to the following optimal concentrations: LtEc(Fe2+) = 0.004 mM heme, metLtEc(Fe3+) = 0.018 mM heme, HbA(Fe2+) = 0.025 mM heme, and metHbA(Fe3+) = 0.055 mM heme. Stocks of 5000× SYPRO orange dye (ThermoFisher Scientific, Waltham, MA, S-6650) were diluted to 200X with Ringer’s solution (115 mM NaCl, 0.3% sodium lactate, 12.25 mM N-acetyl-L-cysteine, 1.4 mM CaCl2, 4.0 mM KCl). About 45 μL of each sample was then mixed with 5 μL of 200× SYPRO orange dye and the thermal shift assay was run on an Applied Biosystems (Waltham, MA) 7300 real-time PCR system that measured sample fluorescence as the temperature was increased from 20 °C to 89 °C in 1 °C increments. The melting temperature (Tm) of each sample was defined as the point of inflection in the fluorescence curve.
Binding affinity in drug design: experimental and computational techniques
Published in Expert Opinion on Drug Discovery, 2019
Visvaldas Kairys, Lina Baranauskiene, Migle Kazlauskiene, Daumantas Matulis, Egidijus Kazlauskas
Interestingly, protein Tm shift upon ligand binding can be also exploited to evaluate target engagement in cells. In the recently discovered cellular thermal shift assay (CETSA [35],), various complex protein samples, including tissues, are aliquoted with and without ligand and subjected to different temperatures. Soluble fractions are isolated from the lysates and quantified using antibody-based assays or mass spectrometry (MS). This approach allows to evaluate ligand efficacy in the desired environment and is being rapidly developed [36]. However, one has to keep in mind that the data is heavily influenced by off-target binding and, in some setups, membrane permeability and thus should not be considered as actual affinity.