Biochemistry instrumentation offers access to techniques invaluable in determining purity, stability, size, conformation and binding characteristics of biomolecules (proteins, DNA, low molecular weight ligands and assemblies). Many of the strategies available are 'in solution', free of interactions with additional matrices, surfaces or labeling and, therefore, better suited to understanding biological processes.
ACQUITY Ultra Performance LC® (UPLC®), High Performance LC (HPLC) and Preparative Liquid chromatography: The ACQUITY UPLC system from Waters Corporation takes advantage of small particle chemistries (1.7µm), ultra-low system volume, low volume detectors and high pressures (15000 psi) to improve resolution, sample throughput and sensitivity for reverse-phase chromatography analysis. The UPLC system is equipped with a photodiode array, fluorescence detection, electrochemical detection and an autosampler. The Waters 625 analytical HPLC system is designed for sensitive analytical methods and is equipped with a photodiode array detector and an autosampler. It is the instrument of choice for separations other than reverse phase.
Size Exclusion Chromatography Multi-Angle Laser Light Scatter (SECMALLS): Fractionation of samples is performed by a Varian Pro-Star system over a gel filtration (sizing) column and delivered to three detection systems: UV/Visible, Refractive Index (RI) and Light Scattering (LS). The Wyatt Dawn Heleos and Wyatt QELS Light Scattering detector is capable of collecting both static and dynamic light scattering data. Static light scatter can directly determine the absolute molar mass, molecular root mean square (rms) radius size and aggregation state. Dynamic light scatter is capable of measuring a protein’s diffusion coefficient, calculating its hydrodynamic size and estimating its molecular weight. Static and Dynamic light scatter methods collect different types of information, providing complementary data for a more complete characterization of biomolecules.
Analytical Ultracentrifugation: The Beckman Optima XL-I is equipped with both absorbance and interference optics, which provides two complementary techniques/approches: Sedimentation Velocity (SV) and Sedimentation Equilibrium (SE). Sedimentation velocity is a hydrodynamic technique sensitive to the mass and shape of the macromolecular species, it can be used to determine sample purity, determine sedimentation coefficients and characterize assembly and disassembly of biomolecular complexes. Sedimentation equilibrium is a thermodynamic technique that is sensitive to the mass but not the shape of the macromolecular species. It can be used for determining molecular mass and equilibrium constants for self- and hetero-associating systems. Experiments are based on first principles and performed in solution. There are no standards or calibrations and no interactions with matrices or surfaces. Experiments can be conducted over a wide range of concentration, temperature, ionic strength and pH.
UV/VIS Spectroscopy and Fluorescence Spectroscopy: The Beckman DU640 UV/VIS spectrophotometer is equipped with software packages for measuring/detecting proteins, nucleic acids and performing kinetics. It also has peltier temperature control and several accessories including microcells for small volumes. Perkin-Elmer’s model LS-50B and Horiba Jobin Yvon’s Fluoromax-3 fluorescence spectrophotometers provide the ability to accurately measure emission and excitation spectra. Options include total emission, polarizers, a 96 well plate reader and temperature control.
Dynamic Light Scattering: The Wyatt DynaPro NanoStar characterizes proteins and other macromolecules by measuring the hydrodynamic radius (ranging from 0.5nm to 1000nm) and the diffusion coefficient. It has a Peltier-driven temperature control capable of temperatures from 0ºC to +90ºC (+/- 1.0ºC). This laser technique is based on the measurement fluctuation in scattered light intensity caused by the relative movements of macromolecules in solution. The DynaPro DLS is capable of measuring small volumes of proteins and is an indispensable tool for biomolecular sizing and characterization, the sample is unaltered and fully retrievable. Monomodal and bimodal regression analysis of the data can be performed.
Circular Dichroism Spectroscopy: CD measurements are performed on a JASCO J-715 spectropolarimeter in the wavelength range of 178-800nm. The instrument is equipped with Peltier temperature control. Experiments include secondary and tertiary structure determination, protein ligand binding and thermal melt studies.