The Spectroscopy and Biophysics Core Facility provides instrumentation, training, and support for any type of experimental work involving biophysical and spectroscopic measurements. While most of the facility’s instruments are configured for use in protein and other macromolecule characterization work, the facility’s services can be extended to partially purified proteins, whole cells with and without media assays, tissues, and small molecules.
Current instrumentation includes:
- Agilent 7500 cx inductively coupled plasma mass spectrometer (ICP-MS) with a 96-well autosampler handling as little as 100 uL of diluted sample per injection, and a total of 20 elements (not including C, H, N, O, F, and Si) can be quantitated in a single injection;
- Agilent LC1200 high-performance liquid chromatograph with diode array detector, also used in conjunction with a QqQ Hybrid mass spectrometer;
- Hi-Tech (Tgk Scientific) stopped-flow rapid kinetics instrument with absorbance, fluorescence, and chemical quench that is also capable of diode-array detection for absorbance and double-mixing for the generation of reaction intermediates;
- Microcal (Malvern) differential scanning calorimeter (DSC) for thermal denaturation studies (range from 10-130 oC) with each compartment having a volume of 0.7 mL (the protein concentration must be below 1 mg/ML);
- Microcal (Malvern) isothermal titration calorimeter (ITC) for the study of macromolecule-ligand or macromolecule-macromolecule binding processes (the cell compartment requires 1.50 mL of target solution at >10 uM concentration and 0.5 mL of ligand or binding partner solution at >200 uM to ensure saturation);
- Varian-Agilent Cary Eclipse spectrofluorimeter with multiple adaptors for cell holders, thermostating, and 96-well plates (wavelength range from 190 to 1100 nm) for high throughput assays at room temperature;
- Beckman XL analytical ultracentrifuge (sedimentation velocity and sedimentation equilibrium can be determined on this instrument without prior knowledge of the system properties, monitored by either absorbance or light interference modes);
- Jasco J-815 circular dichroism equipped with fluorescence and fluorescence polarization accessories as well as stopped-flow for the kinetic study of unfolding processes.
Core services include:
- Determination of protein stability and thermodynamic parameters for protein conformational changes by DSC;
- Ligand-protein and protein-protein binding studies by means of isothermal microcalorimetryor fluorescence anisotropy;
- Stopped-flow spectrophotometry and chemical quench (dead time of 25 ms);
- Fluorescence, phosphorescence, and chemiluminescence experiments in spectral and kinetic modes;
- Elemental analysis by ICP-MS;
- Ligand binding and protein-protein kinetics using SPR technology;
- Analytical ultracentrifugation to determine protein size and protein aggregation;
- Intact mass analysis of proteins by LC-MS;
- Bottom-up proteomics analysis of purified and semi-purified proteins;
- Targeted metabolite analysis using QqQ mass spectrometry;
- Metabolite profiling using QqQ mass spectrometry with statistical analysis.
