Protein Expression and Purification Core Facility

PEPCF expresses proteins in bacteria, insect and mammalian cells and uses a variety of chromatographic and biophysical techniques for protein purification and characterization.


Circular dichroism (CD) spectroscopy

Used for: probing the secondary structure of chiral macromolecules and assessment of conformational stability, structural changes and complex formation by measuring the differential absorption of left and right-handed circularly polarized light. CD is also commonly used for protein quality control.

Wavelength range of our Jasco J-815 instrument: 190-900 nm.

Sample requirements: 80 µl of 0.5-2 mg/ml of sample. Sample buffer should be compatible with CD measurements (buffer components absorbing in the region of interest should be avoided).

Fluorescence spectroscopy

Used for: probing structural changes (folding, assembly, stability) in macromolecules and assessment of macromolecular interactions by measuring the intensity of emitted photons from a sample after it has absorbed photons. It is an in-solution method that makes use of intrinsic fluorophores (Trp, Tyr, Phe) or fluorescent dyes.

Fluorescence polarization is the phenomenon were the light emitted by a fluorophore has unequal intensities along different axes of polarization. This can be used to measure the binding of relatively small fluorescent molecules to their larger target molecule. It is an in-solution method that makes use of fluorescent dyes.

Wavelength range of our Jasco FP-8500 instrument: 200-750 nm.

UV-Vis spectroscopy

Used for: measuring the absorbance spectrum of molecules, determining the concentration of molecules and monitoring enzymatic reactions. It measures a molecule’s light absorption at different wavelengths.

Wavelength range of our Agilent Cary60 instrument: 190-1100 nm.

Agilent Cary60 UV-Vis Spectrometer