Spectroscopy

Director:
Javier Seravalli, PhD
E157 Beadle Center
402-472-3124 (office)
jseravalli1@unl.edu
 

Facilities: Spectroscopy and Biophysics Core

The role of the Spectroscopy and Biophysics Core is to provide instrumentation, training and support for any type of experimental work involving biophysical and spectroscopic measurements. Although most of the instruments listed below are configured for use in protein characterization work, the services that our core provides can be extended to partially purified proteins, whole cell assays and analysis of tissues. The instrumentation is located in rooms E155 and N113B of the Beadle Center and includes an Inductively Coupled Plasma Mass Spectrometer (Agilent 7500 cx), an HPLC with diode array detector (Agilent LC1200), a 96-well plate autosampler (Elemental Scientific Inc), a Stopped Flow rapid kinetics instrument (Hi Tech), a differential scanning calorimeter (Microcal GE Healthcare), a isothermal calorimeter (Microcal GE Healthcare) and a Spectrofluorimeter (Varian, Cary Eclipse).

University of Nebraska Medical Center - Core Facilities and Shared Resources

Services Offered by the RBC Spectroscopy Facility:

  • Determination of protein stability and thermodynamic parameters for protein conformational changes by means of differential scanning calorimetry (DSC). During a DSC experiment, the temperature of the sample cell and a reference cell is ramped up - usually in a linear fashion, and the difference in heat that is necessary reach that temperature is continuously monitored. The heat measurement is translated into enthalpies of transitions and heat capacities.

  • Ligand-protein and protein-protein binding studies by means of isothermal microcalorimetry. A titrator syringe adds a concentrated solution to a sample of protein. The heat evolved or taken during the binding process or a chemical reaction is monitored at a constant temperature set by the user. After correction for heat of dilution of the titrant the enthalpy of binding can be accurately estimated. Studies at several temperatures allow the measurement of entropies and free energies ligand-macromolecule interactions and macromolecule-macromolecule interactions such and protein-protein and protein-DNA complexes.

  • Stopped flow spectrophotometry. Rapid mixing can be achieved in 0.2 milliseconds. Measurements in absorbance, fluorescence and chemiluminescence are routinely performed with detection in single wavelength and diode array (512 nm per scan) configurations. Accessories included are double mixing experiments, a chemical quench 6 port valve (Q-pod) and nitrogen gas attachments for anaerobic experiments

  • Standard Spectrofluorimeter equipped with up to 6 cuvets, a 96-well plate holder and a Peltier thermostated single cuvet holder for fluorescence, phosphorescence and chemiluminescence experiments in spectral and kinetic modes.

  • Elemental Analysis by means of Inductively Coupled Plasma Mass Spectrometry. Almost any element of the periodic chart can be determined down to the parts per trillion (ng per kg or picomolar) concentration except C,H, O, N, F, Cl and noble gases. Samples can be as small as 100 microliters and can be in either liquid or solid states. An HPLC unit is coupled with this instrument which allow the separation of elements in distinct chemical environments (chemical speciation) or the reduction of interferences by reverse phase or ion chromatography. In addition, isotope ratio and spectral semi-quantitative analysis can be performed.

  • Analytical Ultracentrifugation. Determination of the protein quaternary structure of proteins using equilibrium analytical ultracentrifugation of protein solutions in water and deuterium oxide.
     

  • NEW!  JASCO CD spectrometer Model J815.  This spectrophotometer is routinely used to record Circular Dichroism (CD) spectra of Biomolecules in solution, but it can also be used for studies of protein, peptide and protein/ligand complex stability, rapid (stopped flow) conformational changes, fluorescence excitation and emission spectra, fluorescence-detected CD, fluorescence polarization/anisotropy and time and/or temperature dependent studies of these phenomena.  The instrument is equipped with detection between 167 and 900 nm, monochromators for both excitation (polarized light) and emission, Peltier thermal sample holder (10 to 90 C) and a stopped flow for monitoring rapid CD-changes.

Summary of Equipment in the Spectroscopy Core Facility:

 

Agilent ICP-MS 7500cx

Agilent ICP-MS 7500cx with the autosampler shown in the background.

ESI Autosampler

ESI autosampler can handle up to 576 samples per run.

 

 

Cary Eclipse Spectrofluorimeter

Cary Eclipse Spectrofluorimeter with 96-well plate holder in place.

Hi-Tech Stopped flow

Hi-Tech Stopped flow showing the sample handling unit, monochromator, photomultiplier power supply and diode array detector.

 

 

Microcal Differential Scanning Calorimeter and ancillary computer.

Microcal Differential Scanning Calorimeter and ancillary computer.

Microcal Isothermal Calorimeter and computer.

Microcal Isothermal Calorimeter and computer. The syringe titrator is not shown for clarity.

Equipment not pictured

The Spectroscopy and Biophysics Core recently acquired a JASCO CD spectrometer Model J815.  This spectrophotometer is routinely used to record Circular Dichroism (CD) spectra of Biomolecules in solution, but it can also be used for studies of protein, peptide and protein/ligand complex stability, rapid (stopped flow) conformational changes, fluorescence excitation and emission spectra, fluorescence-detected CD, fluorescence polarization/anisotropy and time and/or temperature dependent studies of these phenomena.  The instrument is equipped with detection between 167 and 900 nm, monochromators for both excitation (polarized light) and emission, Peltier thermal sample holder (10 to 90 C) and a stopped flow for monitoring rapid CD-changes.