Is my protein soluble, at the correct concentration and molecular size?
UV –VIS spectroscopy By scanning samples in the range between 320-210nm it is possible to obtain an estimate of protein concentration if the protein contains aromatic amino acids and the extinction coefficient is known. By measuring the absorption ratio at 260/280nm we can establish whether or not nucleic acid contamination is an issue, in addition looking at the baseline around 310nm can give an indication of the presence of light scattering species.
Analytical Ultracentrifugation (AUC) is used to measure sample molecular weight, purity and oligomeric/hydrodynamic state. It separates species in solution, and can be useful to monitor conformational changes. The sedimentation rate of the molecules is induced by centrifugal force to produce a size distribution profile of the species in the sample. Samples can be proteins, nucleic acids, carbohydrates, polymers and complexes of biological molecules. If binding occurs, the affinity and stoichiometry can be determined.
Size Exclusion Chromatography- Multi Angle Light Scattering (SEC-MALS) can be used to tell us the absolute molecular mass, the oligomeric state and the presence of heterogeneous species. It is an ideal tool for quality control measurements prior to any downstream applications.
Dynamic Light Scattering can quickly assess the aggregation of your sample, and determine the hydrodynamic radius.
AUC Sample Criteria
A series of concentrations can be used to monitor the oligomeric state dependence on concentration. Analysis can be multiplexed using 3 or 7 sample holders per run which allows the measurement of up to 28 samples (at different concentrations).
Sample concentrations:
Absorbance optics approx. 0.1-10.0 mg/ml in 12 mm path length cell wavelength 180-800nm.
Interference optics typically 0.05-30mg/ml.
MALS Sample Criteria
Minimal requirements are 25ug of 1mg/ml solution and 500ml of buffer. All samples and buffers must be free from particulates and should be prepared using HPLC grade quality water. All samples and buffers must be filtered immediately prior to use.
Analysis typically takes 2 hours