For each well, 1 μL of 800 μM FDA-approved Drug Library (Selleck Chemical, Houston TX) in 100% DMSO was added to 19 μL of assay buffer in assay plate (Corning 3575) using a Hamilton MicroLab. This was followed by additions of GST-CaM and Cy5-labeled peptide, and FP was measured as described above. Optically interfering compounds were identified by performing absorbance wavelength scan every 5 nm from 350 to 700 nm on BioTek Synergy 2 (Winooski, VT).
Fda approved drug library
Manufactured by Selleck Chemicals
Sourced in United States
The FDA-approved Drug Library is a collection of chemical compounds that have been approved by the U.S. Food and Drug Administration (FDA) for use as drugs. This library serves as a valuable resource for researchers and scientists in the pharmaceutical and biomedical fields.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Digitonin, by Merck Group (1 mentions)
Lps o55 b5, by Merck Group (1 mentions)
7 amino 4 methylcoumarin amc, by Merck Group (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Hrp conjugated sheep antibody against mouse igg, by GE Healthcare (1 mentions)
Hrp conjugated donkey antibody against rabbit igg, by GE Healthcare (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Polyjet, by SignaGen (1 mentions)
Nimesulide, by Selleck Chemicals (1 mentions)
Darolutamide, by Selleck Chemicals (1 mentions)
Epi 001, by Selleck Chemicals (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Crystal violet dye, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Natural product library, by Selleck Chemicals (1 mentions)
10 protocols using fda approved drug library
1
Fluorescence Polarization Assay for Compound Screening
2
Proteolytic Activity Assay Protocol
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The Ac-WEHD-AMC was obtained from Enzo-life science (Farmingdale, NY, USA). The FDA-Approved drug library was obtained from Selleckchem (Houston, TX, USA). β-mercaptoethanol (2ME) and Tween20 were obtained from Bio-Rad (Hercules, CA, USA). The LPS-Ra mutant, LPS O55:B5, Digitonin, DMSO, 7-Amino-4-methylcoumarin (AMC), and all other chemicals were obtained from Sigma-Aldrich (Seoul, South Korea) unless stated otherwise.
3
High-Throughput Screening of FDA-Approved Drugs
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FDA-approved drug library (cat# L1300, 100 µl/well, 10 mM solution in DMSO) was purchased from Selleck Chemicals LLC and used for high-throughput screening in this study. The glucocorticoid receptor antagonist, RU-488, and dexamethasone used in this study are from this library. Hydrocortisone and CCCP were purchased from Sigma. The following primary antibodies were used: mouse monoclonal antibody against parkin (cat# 4211, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against pPERK (cat# 3179, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against PERK (cat# 5683, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against CREB (Cat# 9197, 1:5,000, Cell Signaling Technology), and rabbit polyclonal antibody against AIMP2 (cat# 10424-1-AP, 1:5,000, Proteintech). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated sheep antibody against mouse IgG (cat# RPN4301, 1:5,000, GE Healthcare), HRP-conjugated donkey antibody against rabbit IgG (cat# RPN4101, 1:5,000, GE Healthcare), and HRP-conjugated mouse antibody against β-actin (cat# A3854, 1:10,000, Sigma-Aldrich).
4
Screening FDA-Approved Drugs on T Cells
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293 T cell lines were plated into 6-wellplate and grown in DMEM (Gibco) supplemented with 10% FBS (Invitrogen), 1% penicillin/streptomycin(Gibco) at 37 °C in 5% CO2 Transient transfections with PolyJet (SignaGen) according to the manufacturer's instructions. The reactions were terminated and immunoblotted with anti-Cdc7 (Thermo-Fisher) and Anti-Dbf4 (LTK BioLaboratories [27 (link)],). Libraries used are: 1175 FDA-approved drug library (SelleckChem, L1300-01). FDA approved drugs supplied as pre-dissolved DMSO solutions.
5
Evaluating Anticancer Drug Combination
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The drugs nimesulide (Oficinal Pharmacy, São Paulo, SP, BR), prednisolone (Biossynthetic, São Paulo, SP, BR), and cytarabine (Libbs, , São Paulo, SP, BR) were used in the cellular experiments. nimesulide was diluted in dimethyl sulfoxide (DMSO) in a 10-mM stock solution, and prednisolone and cytarabine, presented in liquid form, were diluted in RPMI medium immediately before being applied to the cells. Food and Drug Administration (FDA)-approved drug library (Selleck Chemicals, Houston, TX, USA) was used for drug screening in combination with nimesulide.
6
Comprehensive Drug Library Preparation
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Enzalutamide (MCE, HY‐70002, NJ, US), EPI‐001 (Selleck, S7955, TX, US), Auranofin (MCE, HY‐B1123, NJ, US), Polaprezinc (MCE, HY‐B0729, NJ, US) and Darolutamide (Selleck, S7559, TX, US) were stored as stock solutions in DMSO (Sigma, MO, US). FDA‐approved drug library (Selleck, L1300, TX, US) was stored at −80 °C.
7
Evaluation of FDA-Approved Drug Library
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The FDA-approved drug library was purchased from Selleckchem (Houston, TX, USA). Crystal violet dye, RPMI 1640 medium, and propidium iodide were purchased from Fisher Scientific (Hampton, NH, USA). Proflavine hemisulfate hydrate was purchased from TCI Chemicals (Tokyo, Japan). All cell lines were purchased from ATCC (Manassas, VA, USA) and maintained in recommended medium supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone) and 1% (v/v) penicillin/streptomycin.
8
Screening for SARS-CoV-2 Main Protease Inhibitors
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Potential inhibitors against SARS-CoV-2 M pro were screened by an enzymatic inhibition assay. When the different compounds were added into the enzymatic reaction mixture, the change of initial rates was calculated to evaluate their inhibitory effect. Five drug librariesthe Approved Drug Library (Target Mol), Clinic Compound Library (Target Mol), FDA-approved Drug Library (Selleck), Natural Product Library (Selleck), and Anti-virus Drug Library (Shanghai Institute for Advanced Immunochemical Studies)-that together comprised about 10,000 compounds were used. The preliminary screening reaction mixture included 0.2 μM protein, 20 μM substrate and 50 μM compounds. The compounds of interest were defined as those with a percentage of inhibition over 60% compared with the reaction in the absence of inhibitor. IC 50 values of 7 drug leads were measured using 0.2 μM protein, 20 μM substrate and 11 different inhibitor concentrations. To exclude inhibitors possibly acting as aggregators, a detergent-based control was performed by adding 0.001% or 0.01% freshly made up Triton X-100 to the reaction at the same time 25 . All experimental data was analysed using GraphPad Prism. All experiments were performed in triplicate.
9
High-Throughput Screening of FDA and Pandemic Drugs
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The primary screen in this study used both the Selleck Chem FDA-Approved Drug Library (#L1300, 1,591 compounds) and the Medicines for Malaria Venture Pandemic Response Box (399 compounds). Details about the sources of drugs for subsequent assays can be found in File S2.
10
High-Throughput Screening of Raf-PHB Interaction
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The experiment was performed as described previously [29] . Brie y, the rst layer antibody His-tag antibody (Proteintech) in coating buffer (1 ng/μL) was added to a 96-well plate and cultured overnight at 4°C. After washing with PBS and blocking with 5% bovine serum albumin (BSA), the puri ed fusion protein Raf-His (1 μg) was added, and the plate was softly rocked for 5 h at room temperature. The plate was then incubated with 1 μg puri ed PHB-GST protein for 3 h at 37°C, and 440 small molecular inhibitors (10 μM) from the FDA-approved Drug Library (Selleck Chemicals, USA) were added individually into each well. After incubation with GST-tagged antibody, the corresponding secondary antibody and tetramethylbenzidine (TMB), the absorbance was measured at wavelengths of 450 nm and 630 nm. The level of Raf-PHB interaction was determined using the formula O.D.450-O.D.630. Coating buffer, TMB and termination buffer were purchased from NeoBioscience (Shenzhen, China).
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