Protein expression and purification can present significant challenges. For this reason, from the early stages of a project, extensive analysis and characterization of your protein is essential to establish a well-characterized molecule. Nexelis offers a wide variety of physicochemical analyses using state-of-the-art instrumentation to understand protein structure, identity, purity, activity, immunochemical and physicochemical properties.
A sensitive method to investigate the structure of proteins. The aromatic amino acid residues exhibit fluorescence emission when excited in the wavelength range of their absorption spectra. However, the emitted fluorescence is usually dominated by the contribution of the tryptophan and subtle structural changes in the tertiary protein conformation under different conditions (temperature, pH, ionic strength, denaturant) can be assessed.
Differential Scanning Fluorimetry (DSF)
DSF is a thermal shift assay to measure the temperature at which a protein goes from a well-defined structure to disorder by analysis of the thermal denaturation melting curve. It is also useful for construct stability comparison, to identify optimal buffer conditions, and batch-to-batch Tm validation.
Near Ultraviolet Absorbance Spectroscopy Second Derivative (UV)
The second derivative of an absorbance spectrum to monitor signals from the three aromatic amino acids in a protein enhances resolution and spectral overlap is significantly reduced. By monitoring the individual shifts of the derivative peak position of these residues, information can be obtained regarding conformational alterations of proteins as a function of conditions, such as pH, temperature, and ionic strength.
Glutaraldehyde is a linear 5-carbon dialdehyde that reacts primarily with amine groups, but also with several other functional groups, such as thiol, phenol and imidazole and allows the fixation of the quaternary structure of the protein in solution. Combined with SDS-PAGE, glutaraldehyde cross-linking is a structural orthogonal method to visualize differences in protein migration before and after treatment.
Circular dichroism (CD) spectroscopy provides information based on the ability of a sample to absorb differently right circularly polarized light and left circularly polarized light. CD is a sensitive tool that provides information on the secondary structure composition (α-helix, β-sheet, β-turns and random coil) based on absorption by peptide bonds in the far-UV CD and on the tertiary structure based on the absorption by either aromatic amino acids and disulphide bonds in the near-UV. CD is mainly used for monitoring structural changes induced by ambient pH, denaturants and temperature.
Protein deglycosylation analysis
Glycosylation is one of the most common protein post-translational modifications and often results in heterogeneity in the mass and charge of glycoproteins. A large amount of information with respect to the structure of a glycoprotein can be obtained by removing N-linked oligosaccharides using enzymatic deglycosylation. The difference in protein migration on SDS-PAGE before and after glycosidase treatment can provide valuable evidence about the glycosylation state of a protein of interest.
Differential Scanning Calorimetry (DSC)
DSC is used to assess protein stability in the native state that directly measures the transition midpoint (Tm) of thermally induced structural transitions without the use of extrinsic dye. DSC monitors thermal unfolding events by measuring changes in apparent excess heat capacity of protein samples during temperature up-scan at constant pressure.
Sedimentation Velocity Analytical ultracentrifugation (SV-AUC)
An analytical ultracentrifugation method measures the rate at which molecules move through their buffer in response to centrifugal force. This sedimentation rate provides information about both the molecular mass and shape. Analytical ultracentrifugation (AUC) separates protein species directly in solution, without the use of a stationary phase such as in size-exclusion chromatography (SEC).
Dynamic light scattering (DLS)
Dynamic light scattering (DLS) is used to determine the hydrodynamic radius distribution of the protein through measurement of the fluctuations in the scattered light intensity and the calculation of the translational diffusion coefficient directly in the native protein condition. Hence, the DLS can assess the level of oligomeric state and the population distribution of protein.
Size-Exclusion Chromatography with Multi-Angle Laser Scattering (SEC-MALS)
MALS technology, in combination with size-exclusion chromatography, measures the absolute molar mass of each peak, independent of the molecular shape or elution position of sample fractions, making SEC-MALS an excellent method for the determination of the size of a protein or complex in solution.
Electron microscopy (TEM)
A technique for obtaining high resolution images of biological samples after negative staining. Images will be taken at the proper magnification.
Peptide mapping by Mass Spectrometry
Peptide mapping is used for identity confirmation by providing primary amino acid sequence confirmation as well as the identification and quantification of post-translational modifications (PTMs) and monitoring degradative events after cleavage of the protein into proteolytic peptides.
Affinity Measurement and Biomolecular Interactions Analysis
Bio-layer interferometry (BLI) biosensor platform, developed by FortéBio, is a real-time label-free detection system for molecular interaction analysis, based on the principles of optical interferometry. BLI measures biomolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. The BLI platform can be used for protein/antibody quantitation, for affinity and kinetics analysis and to determine the binding affinity constant (KD).