We pride ourselves on the plethora of technologies we use within our laboratories to address client projects. Having expertise in the technologies listed below, allows us to offer to each client an opportunity to prosecute their target to the best of our capabilities.
Implementation of each technology at a specific stage of the screening cascade, based on throughput at one end of the scale or physiological relevance at the other, allows us to tailor our solutions to client discovery programs.
SPR can be used in the initial stages of the project to examine binding of compounds to the target protein. This approach provides binding kinetics (on / off rates) of compounds binding to the protein; however, SPR requires purified protein and an appropriate chemical handle to link the protein to the SPR chip.
This approach can be used for fragment-based drug discovery, to begin to elucidate the binding site properties of the protein and in combination with Computer-Aided Design (CAD), allows for modelling of the protein binding pocket.
By using a qPCR instrument, we can examine the temperature related unfolding of a protein.
Using a fluorescence dye that incorporates into the hydrophilic core of the protein and effectively “cages” (blocks) the fluorescence, as the temperature of the environment increases the protein unfolds, the dye becomes “visible”, and is able to emit fluorescence.
The binding of a compound to the protein stabilizes the protein structure, therefore the protein unfolds at a higher “melt” temperature. The degree of difference between native protein and compound bound plus protein indication, indicates that a compound has bound and stabilized the protein.
Typically for client projects we purchase “Cell to CT” kits and design primers adapted for this approach. In either a siRNA knockdown or TPD project, we examine the kinetic expression of message and compound concentration using our 384 well qPCR instrument for maximum throughput. We use a loading control mRNA in all lysates to ratio the target mRNA to a mRNA species not affected by the treatment.
Traditional western blot is used to examine the presence and concentration of a specific protein in a cell lysis.
This technique has remained constant since its introduction in the 1970’s. The Simple western system is an automated western blot technology using columns established by the instrument in glass capillaries rather than gels.
The instrument can screen 24 lysates in each run which takes between three and four hours to complete. Multiple proteins can be detected in each lane, separated on the based on molecular weight, and readouts can include both luminescence (using horse radish peroxidase) or near-infra-red or infra-red for say total and phospho protein detection. This technology has been extensively used by our clients for Targeted Protein Degradation and Protein Stabilization projects.
By using glass beads, this instrument can effectively “pulverise” tissues ranging from liver, brain, muscle, bone etc to form a cell lysate that can then be used for western blotting studies. This approach has been used by clients to examine knockdown of proteins in-vivo by either siRNA or TPD molecules.
FLIPR is a kinetic imaging plate read that, combined with the use of fluorescence dyes, can examine the movement (flux) of calcium and potassium from compartments within cells that are adhered to the base of the microwell plate.
Containing a CCD camera and LED excitation light sources plus emission filters, the instrument can image all wells of the plate simultaneously, and take images of all wells rapidly, every 0.3 seconds or longer, allowing for kinetic reads of every well simultaneously. C
ells are incubated with cell permeable fluorescence dye that, when calcium is released from internal cell stores, binds to the calcium and becomes fluorescent. Calcium is normally released upon activation of many G-Protein Coupled Receptors (GPCR’s). Activation of the GPCR by binding of a ligand, results in calcium mobilization. The FLIPR instrument also contains a multi-channel dispensing head (either 96 or 384 well), allowing for dispensing of both the activating ligand and the inhibitor compound during a kinetic read.
We have several plate readers from different manufacturers including PerkinElmer and BMG.
Each has different properties:
- With and without stackers for HTS purposes
- Monochromator or filter-based
- Different plate formats including 96, 384 and 1536 well plates
- Label-free assay format (Corning EPIC technology)
- Enhanced luminescence in addition to prompt fluorescence, HTRF, AlphaLISA, Fluorescence Polarization, BRET.
We also have a plate reader with a heated stage read position that is sparged and CO2 to allow for kinetic plate based assays. This has allowed us to use CRISPR knock-in cell lines from Promega, combined with long lived Endurazine substrate, to examine PROTAC and molecular glue activity over a 24-hour kinetic read in which expression of the target protein is under endogenous cellular control. The rate of recovery of degraded proteins is as important and the rate of degradation following TPD.
Significantly used by clients for blood-based cytokine assays, this approach is used in combination with our human blood donor panel for PBMC, T- and B-cell assays.
By using bead-based readouts, we can multiplex numerous cytokine measurements to accurately determine the response of donor blood cells. In addition, we use flow cytometry to examine cell surface receptor expression and, when the cells have been permeabilized, we are able to examine fluorescence antibody labelled staining inside the cell, which is often used for suspension rather than adherent cells because of the simplicity of the staining procedure with non-adherent cells.
We have several lasers within each of our instruments allowing us to examine several different fluorophores simultaneously within the same sample.
Incucyte is much more than a microscope in box within the incubator. The power of the technology lies in the software, allowing for numerous cell-based applications, and can be combined with specialist application-based microplates.
Our imagers have a 4x, 10x and 20x magnification optics combined with several excitation wavelengths that allow us to study cellular events in real time kinetics, taking images of the cells and applying software algorithms to convert these images to data.
Specialized microplates allow us to study chemotaxis, phagocytosis as well as T-cell killing in both 2D and 3D formats.
