Journal: Nature Communications

Article Title: CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

doi: 10.1038/ncomms11040

Figure Lengend Snippet: ( a ) Overview of the assay principle with live K562 cells seeded into a 384-well PCR plate. The plate contains controls or library compounds that are taken up by the cells. Following a pre-incubation period the plate is transiently heated for 3 min followed by cooling and cell lysis. Part of the cell lysate is transferred to a detection plate, to which antibodies and AlphaScreen beads are added to allow measurements of remaining soluble TS. ( b ) CETSA derived T agg curves for TS in K562 cells in the presence of DMSO (0.5%) (green circle), 15 μM floxuridine (blue triangle) or 1 μM raltitrexed (magenta square). All data were normalized to the response observed for each treatment condition at the lowest test temperature. The solid line represents the best fit to the Boltzmann sigmoid equation resulting in an apparent T agg of 46.7±0.2 °C for the DMSO control, whereas both floxuridine and raltitrexed stabilized TS above 65 °C (we do not consider higher T agg values reliable as these temperatures influence cell membrane integrity ). The vertical dotted line is at 50 °C, the temperature selected for the isothermal screen. Data are provided as the average and standard error of mean (s.e.m.) from two independent experiments performed in duplicate for raltitrexed and as individual data points from one experiment in duplicate for floxuridine. ( c ) ITDRF CETSA of floxuridine (blue triangle) at 50 °C based on raw data from the AlphaScreen readings. The solid line represents the best fit to a saturation binding curve resulting in an EC 50 of 47±16 pM. Data are provided as two individual data points from one test occasion. ( d ) The corresponding ITDRF CETSA for raltitrexed (magenta square) at 50 °C resulting in an EC 50 of 0.75±0.2 nM. Data are provided as two individual data points from one test occasion.

Article Snippet: Finally 5 μl of the diluted compounds were transferred to a 384 well hardshell PCR plate (HSR480, BIORAD) using a Bravo liquid handling platform equipped with a 384-well head (Agilent).

Techniques: Incubation, Lysis, Amplified Luminescent Proximity Homogenous Assay, Derivative Assay, Control, Membrane, Binding Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

doi: 10.3791/59495

Figure Lengend Snippet: Table of Materials

Article Snippet: As we unravel the biology underlying its rapid growth, further development of V. natriegens cell- free systems may accelerate bioproduction capabilities and enable robust expression of therapeutic peptides, small molecules, and synthetic materials. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments 15 mL Tubes Corning 352196 2 mL Tubes Eppendorf 22363352 384-well Black Assay Plates Corning 3544 384-well PCR Plates Eppendorf 951020702 50 mL Tubes Corning 352070 96-well PCR Plates Eppendorf 30129300 Adenosine 3',5'-cyclic monophosphate sodium salt monohydrate Sigma A6885 Adenosine 5'-triphosphate - 100 mM NEB N0450L Applied Biosystems Veriti 384-well Thermocycler ThermoFisher 4388444 Assay Plate Adhesives BioRad MSB1001 β-Nicotinamide adenine dinucleotide hydrate (NAD) Sigma/Roche 10127965001 Coenzyme A hydrate Sigma C4283 Cytidine 5'-triphosphate - 100 mM NEB N0450L D-(-)-3-Phosphoglyceric acid disodium salt (3-PGA) Sigma P8877 Dewar Flask - 4L ThermoScientific 10-194-100C DL-Dithiothreitol solution - 1 M Sigma 42816 Folinic acid calcium salt hydrate Sigma 47612 Glacial Acetic Acid Sigma A6283 Guanosine 5'-triphosphate - 100 mM NEB N0450L HEPES Sigma H3375 L-Glutamic acid hemimagnesium salt tetrahydrate Sigma 49605 L-Glutamic acid potassium salt monohydrate Sigma G1149 LB Broth (Miller) Sigma L3522 Magnesium chloride (MgCl2) Sigma M8266 Plasmid pJL1-sfGFP Addgene 69496 Plasmid Plus Maxi kit Qiagen 12963 Poly(ethylene glycol) (PEG)-8000 Sigma 89510 Potassium chloride (KCl) Sigma P9333 Potassium hydroxide Pellets Sigma/Roche 1050121000 Q125 Sonicator and CL-18 probe with a?-inch tip Qsonica 4422 RNA Clean and Concentrator Kit Zymo R1013 RNase Inhibitor, Murine NEB M0314 RTS Amino Acid Sampler Biotechrabbit BR1401801 Sodium chloride (NaCl) Sigma S7653 Spermidine Sigma S0266 T7 RNA Polymerase NEB M0251 Tris Solution (pH 8.0) - 1 M Invitrogen AM9856 tRNA from E. coli MRE 600 Sigma/Roche 10109541001 Uridine 5'-triphosphate - 100 mM NEB N0450L Vibrio natriegens (Wild-type) Lyophilized Stock ATCC 14048 Open in a separate window

Techniques: Plasmid Preparation

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: A multiplexed high throughput screening assay using flow cytometry identifies glycolytic molecular probes in bloodstream form Trypanosoma brucei

doi: 10.1016/j.ijpddr.2024.100557

Figure Lengend Snippet: Parameters for Multiplexed biosensor Small Molecule Screen.

Article Snippet: HTS process , Format , 80 μL 384-well plate (BrandTech 781620).

Techniques: Fluorescence, Flow Cytometry, Drug discovery, Control, Concentration Assay, Positive Control, Incubation, Negative Control, Software, Selection, Comparison, Purification