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Current Protocols in Protein Science Feb 2015Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization...
Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.
Topics: Biological Assay; Fluorometry; Ligands; Protein Stability; Proteins; Temperature
PubMed: 25640896
DOI: 10.1002/0471140864.ps2809s79 -
Current Protocols in Immunology Nov 2018Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at...
Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc.
Topics: Antibodies; Biological Assay; DNA Primers; Humans; Microscopy, Fluorescence; Protein Interaction Mapping
PubMed: 30238640
DOI: 10.1002/cpim.58 -
Cell Adhesion & Migration 2014The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of...
The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.
Topics: Biological Assay; Human Umbilical Vein Endothelial Cells; Humans; Microscopy; Wound Healing
PubMed: 25482647
DOI: 10.4161/cam.36224 -
Sensors (Basel, Switzerland) 2012Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include... (Review)
Review
Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls.
Topics: Animals; Biological Assay; Cell Survival; Coloring Agents; Humans; Indicators and Reagents; Oxazines; Xanthenes
PubMed: 23112716
DOI: 10.3390/s120912347 -
SLAS Discovery : Advancing Life... Jun 2019Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its...
Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.
Topics: 3C Viral Proteases; Biological Assay; Cysteine Endopeptidases; Enzyme Assays; Humans; Research Design; Substrate Specificity; Viral Proteins
PubMed: 30802413
DOI: 10.1177/2472555219830084 -
Journal of Visualized Experiments : JoVE Mar 2018The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell...
The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 μm). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.
Topics: Animals; Biological Assay; Cell Movement; Humans; Wound Healing
PubMed: 29608162
DOI: 10.3791/56825 -
Basic & Clinical Pharmacology &... May 2021Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been... (Review)
Review
Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.
Topics: Biological Assay; Biomarkers; Diagnostic Test Approval; European Union; Pharmacogenomic Testing; Precision Medicine; Reagent Kits, Diagnostic; United States; United States Food and Drug Administration
PubMed: 33665955
DOI: 10.1111/bcpt.13578 -
Natural Product Reports Jul 2020Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product... (Review)
Review
Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product Discovery. As part of this effort, the NCI is producing a library of 1 000 000 partially purified natural product fractions which are being plated into 384-well plates and provided to the research community free of charge. As the first 326 000 of these fractions have now been made available, this review seeks to describe the general methods used to collect organisms, extract those organisms, and create a prefractionated library. Importantly, this review also details both cell-based and cell-free bioassay methods and the adaptations necessary to those methods to productively screen natural product libraries. Finally, this review briefly describes post-screen dereplication and compound purification and scale up procedures which can efficiently identify active compounds and produce sufficient quantities of natural products for further pre-clinical development.
Topics: Biological Assay; Biological Products; Drug Discovery; High-Throughput Screening Assays; Humans; Small Molecule Libraries
PubMed: 32186299
DOI: 10.1039/c9np00068b -
Genes Oct 2020Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical...
Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3'-P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3'-P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.
Topics: Amino Acids; Amino Acyl-tRNA Synthetases; Biological Assay; Humans; RNA, Transfer; Transfer RNA Aminoacylation
PubMed: 33036365
DOI: 10.3390/genes11101173 -
Bioanalysis Apr 2022To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Mild acidic assay conditions and capture and...
To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Mild acidic assay conditions and capture and detection antibodies with different affinities and t under different assay pHs were used to mitigate interference in the total drug and total target assays. A free target assay was also developed using a lower-affinity capture antibody with a much slower association and dissociation rate. The impact of sample incubation, dilution and storage on the accurate detection of the free target was also evaluated. The total drug, total and free target assays can accurately quantitate drug and target concentrations when tested with a subset of clinical study samples.
Topics: Antibodies, Monoclonal; Biological Assay; Indicators and Reagents
PubMed: 35297286
DOI: 10.4155/bio-2021-0276