BMG ClarioStar Plate Reader: Our top-of-the-line plate reader has fluorescence, luminescence, absorbance, and fluorescence polarization modes using either filters or monochromators. Additionally, two syringes allow for injection into 96-well or 384-well plates for performing time-controlled additions with simultaneous reads.
Opentrons Pipetting Robot: This liquid handling robot (named ‘Otto’) allows for automation of medium to high throughput assays. We use this technology for buffer and inhibitor screens as well as serial dilutions. It is equipped with both multi-channel and single channel pipette heads, which allow for pipetting volumes from 1 to 300 ul in everything from Eppendorf tubes to 384-well plates.
Mass Photometer: Mass photometry is a new method of analyzing molecules with light. It allows the accurate mass measurement of single molecules in solution, in their native state and without labels. Our new Refeyn TwoMP is located in-house. We are currently using mass photometry to determine the stoichiometry of macromolecular complexes, observe protein oligomerization, and assess heterogeneity of protein:protein and DNA:protein samples.
Analytical Ultracentrifugation: Analytical ultracentrifugation has become the method of choice to analyze structural transitions in chromatin. The method is based on first principles and allows a wide variation of solute conditions. Information on molecular weight, shape, and sample homogeneity can be obtained, using the Ultrascan software packages.
Our Beckmann XLA ultracentrifuge is equipped with an Aviv Fluorescence detection system, allowing us to detect labeled compounds at low nanomolar concentration in a complex mixture.
Atomic Force Microscopy: The Atomic Force Microscope (AFM) is a device used to image materials and biological samples at the single-molecule level. It uses an extremely fine tip positioned at the end of a cantilevered bar. As the tip is moved over the substrate, it periodically taps the surface and bends as it is repelled or attracted to the structure. A laser picks up the deflections, resulting in a direct three-dimensional image of single molecules. Although the resolution of these images does not reach that of structures obtained by x-ray crystallography, a major advantage of this approach lies in the ability to visualize single macromolecules under a variety of conditions.
We are equipped with the JPK Nanowizard ULTRASpeed Atomic Force Microscope which is highly suitable to image and manipulate single biological macromolecules in dry or aqueous environments. The system couples very low noise with fast scan speeds to produce high resolution images in a user-friendly platform. The data we are able to interpret from this instrument includes; the change in the size (height) of the complex when protein(s) are added, the change in overall geometry of the particles and the stability of our complexes. Our AFM is located on site in basement of the JSCBB. Alison White manages this equipment.
ICT: In Isothermal Titration Calorimetry (ITC) heat changes associated with chemical reactions are monitored. It is a powerful, label-free, direct method to observe the thermodynamics of binding reactions and to directly characterize enzyme kinetics. We use isothermal calorimetry (ITC) to determine binding constants of protein:protein and protein:ligand interactions. We have a VP-ITC and an ITC200 instrument from MicroCal. Both instruments can directly measure affinities from the high micromolar to low nanomolar.
Fluorescence Spectroscopy: Fluorescence spectroscopy is widely used in biological sciences, biochemistry, and biophysics. Intrinsic fluorescence (E.g. tryptophans, tyrosines, phenylalanines, co-factors) as well as site directed fluorescence labeling can be exploited to investigate properties of soluble biological molecules (E.g. proteins, nucleic acids, peptides), membranes and macromolecular complexes in solution. We use fluorescence spectroscopy to investigate the thermodynamics of nucleosomes and chromatin using a SX20 fluorescence stopped-flow from Applied Photophysics and HoribaJobinYvon Fluorolog 3.
We have access to two brand-new Typhoon 9500 phosphorimagers; these are used for plate assays as well as for scanning of gels with fluorescent components. The Typhoon is a variable-mode imager that produces digital images of radioactive, fluorescent, or chemiluminescent samples. Automated three-color fluorescence scanning allows multiplexing of multiple targets in the same sample. It can be used to scan phosphor stage screens, gel sandwiches, agarose and polyacrylamide gels, membranes, microplates and even microarrays.
Light Scattering: Wyatt Technologies Dawn Heleos II light scattering device connected to an AKTA HPLC system allows us to analyze the molecular weight of components from complex mixtures, as they elute from a gel filtration column (SEC-MALS). This allows us to rapidly determine the stoichiometry of macromolecular complexes. We use a Wyatt Calypso II for rapid, quantitative characterization of protein-protein and other macromolecular interactions with composition-gradient multi-angle light scattering.
HDX-MS: We use HDX-MS to map protein-protein and protein-DNA interfaces, and to monitor structural changes in chromatin-associated proteins. These experiments are performed in collaboration with Dr. Natalie Ahn and the CU Boulder Mass Spectrometry Facility.
Several pieces of our instruments are available for shared use through the “Biochemistry Shared Instrument Pool”. More information is available at here https://www.colorado.edu/lab/biochem-instruments/