Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
Diamond Light Source is the UK's national synchrotron light source science facility located at the Harwell Science and Innovation Campus in Oxfordshire. Its purpose is to produce intense beams of light whose special characteristics are useful in many areas of scientific research. In particular it can be used to investigate the structure and properties of a wide range of materials from proteins, and engineering components to conservation of archeological artifacts.
Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
High-throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing/control software, liquid handling devices, and sensitive detectors, high-throughput screening allows a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the noninteraction or role of a particular location.
Biological small-angle scattering is a small-angle scattering method for structure analysis of biological materials. Small-angle scattering is used to study the structure of a variety of objects such as solutions of biological macromolecules, nanocomposites, alloys, and synthetic polymers. Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are the two complementary techniques known jointly as small-angle scattering (SAS). SAS is an analogous method to X-ray and neutron diffraction, wide angle X-ray scattering, as well as to static light scattering. In contrast to other X-ray and neutron scattering methods, SAS yields information on the sizes and shapes of both crystalline and non-crystalline particles. When used to study biological materials, which are very often in aqueous solution, the scattering pattern is orientation averaged.
Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. SPR is the basis of many standard tools for measuring adsorption of material onto planar metal surfaces or onto the surface of metal nanoparticles. It is the fundamental principle behind many color-based biosensor applications, different lab-on-a-chip sensors and diatom photosynthesis.
Nanoparticle tracking analysis (NTA) is a method for visualizing and analyzing particles in liquids that relates the rate of Brownian motion to particle size. The rate of movement is related only to the viscosity and temperature of the liquid; it is not influenced by particle density or refractive index. NTA allows the determination of a size distribution profile of small particles with a diameter of approximately 10-1000 nanometers (nm) in liquid suspension.
A particle counter is an instrument that detects and counts physical particles.
High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screen conducted in cells involving the analysis of whole cells or components of cells with simultaneous readout of several parameters. HCS is related to high-throughput screening (HTS), in which thousands of compounds are tested in parallel for their activity in one or more biological assays, but involves assays of more complex cellular phenotypes as outputs. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology of the cell. Hence HCA typically involves automated microscopy and image analysis. Unlike high-content analysis, high-content screening implies a level of throughput which is why the term "screening" differentiates HCS from HCA, which may be high in content but low in throughput.
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed by means of the intensity or photon auto-correlation function. In the time domain analysis, the autocorrelation function (ACF) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. It has been shown that the intensity ACF is the Fourier transformation of the power spectrum, and therefore the DLS measurements can be equally well performed in the spectral domain. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions.
Hit to lead (H2L) also known as lead generation is a stage in early drug discovery where small molecule hits from a high throughput screen (HTS) are evaluated and undergo limited optimization to identify promising lead compounds. These lead compounds undergo more extensive optimization in a subsequent step of drug discovery called lead optimization (LO). The drug discovery process generally follows the following path that includes a hit to lead stage:
Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight Mw of a macromolecule like a polymer or a protein in solution. Measurement of the scattering intensity at many angles allows calculation of the root mean square radius, also called the radius of gyration Rg. By measuring the scattering intensity for many samples of various concentrations, the second virial coefficient A2, can be calculated.
Particle size analysis, particle size measurement, or simply particle sizing is the collective name of the technical procedures, or laboratory techniques which determines the size range, and/or the average, or mean size of the particles in a powder or liquid sample.
Multiangle light scattering (MALS) describes a technique for measuring the light scattered by a sample into a plurality of angles. It is used for determining both the absolute molar mass and the average size of molecules in solution, by detecting how they scatter light. Collimated light from a laser source is most often used, in which case the technique can be referred to as multiangle laser light scattering (MALLS). The insertion of the word “laser” was intended to reassure those used to making light scattering measurements with conventional light sources such as Hg-arc lamps that low angle measurements could now be made. Until the advent of lasers and their associated fine beams of narrow width, the width of conventional light beams used to make such measurements prevented data collection at smaller scattering angles. In recent years, since all commercial light scattering instrumentation use laser sources, this need to mention the light source has been dropped and the term MALS used throughout.
Brookhaven Instruments Corporation is a Nova Instruments company. Established in the late 1960s, Brookhaven Instruments pioneered modern techniques in characterizing nanoparticles, proteins, and polymers using light scattering techniques such as dynamic, static, electrophoretic, and phase analysis for: particle size, zeta potential, molecular mass and absolute molar mass analysis.
There are many methods to investigate protein–protein interactions which are the physical contacts of high specificity established between two or more protein molecules involving electrostatic forces and hydrophobic effects. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are occurring in reality.
Richard Dale Smith is an award-winning chemist and a Battelle Fellow and Chief Scientist within the Biological Sciences Division, as well as the Director of Proteomics Research at the Pacific Northwest National Laboratory (PNNL). Dr. Smith is also Director of the NIH Proteomics Research Resource for Integrative Biology, Principal Investigator for the U. S. Department of Energy Office of Biological and Environmental Research Genome Sciences Program's 'Pan-omics' program at PNNL, an adjunct faculty member in the chemistry departments at Washington State University and the University of Utah, and an affiliate faculty member at the University of Idaho and the Department of Molecular Microbiology & Immunology, Oregon Health & Science University. He is the author or co-author of approximately 1000 peer-reviewed publications and has been awarded 51 US patents.
A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.
NanoSight Ltd is a company that designs and manufactures instruments for the scientific analysis of nanoparticles that are between approximately ten nanometers (nm) and one micron (μm) in diameter. The company was founded in 2003 by Bob Carr and John Knowles to further develop a technique Bob Carr had invented to visualize nanoparticles suspended in liquid. The company has since developed the technique of Nanoparticle Tracking Analysis (NTA), and they produce a series of instruments to count, size and visualize nanoparticles in liquid suspension using this patented technology.
Microfluidic diffusional sizing (MDS) is a method to measure the size of particles based on the degree to which they diffuse within a microfluidic laminar flow. It allows size measurements to be taken from extremely small quantities of material (nano-grams) and is particularly useful when sizing molecules which may vary in size depending on their environment - e.g. protein molecules which may unfold or become denatured in unfavourable conditions.
