Application note details how scientists were able to more rapidly determine Mw and A2 with continuous flow to detectors, by accessorising Calypso automated protein association and aggregation system
Wyatt Technology says its Calypso system has been proven to reduce experimental preparation time and decrease noise levels when measuring the light scattering data of imperfect samples.
Calypso performs rapid, quantitative, non-destructive characterisation of protein-protein interactions in native solution using composition-gradient multi-angle light scattering (CG-Mals).
Normally Calypso operates in stop-flow mode, for optimal sensitivity with minimal sample consumption, and to observe reaction kinetics.
Continuous flow improves the data obtained with difficult or imperfect samples, by moving any large particulate through the system rapidly.
This enables the despiking firmware inherent in Wyatt Technology light scattering instruments to recognise and remove the noise.
In addition to removing data artifacts due to pressure fluctuations, the continuous flow method also reduces build-up of sticky samples, eg, membrane proteins, on the surfaces of detector flow cells.
Besides the standard Calypso automated mixing and dilution hardware, two additional pieces of equipment are required for continuous flow mode: a standard HPLC pump and an 8 port valve.
The dual-sample-loop, eight-port valve is controlled directly by the Calypso software.
Via the user-friendly graphic interface, a two-step method is readily designed: the Calypso syringe pumps prepare the sample and load the sample loop, then a command is issued to the valve to deliver the sample to the detectors.
As the isocratic HPLC pump shoulders the burden of high-pressure flow, wear and tear on the precision mixing syringe pumps is reduced and higher flow rates are supported.
For this particular application CG-Mals data from 40kD dextran were acquired and processed to determine weight-averaged molar mass and second virial coefficient A2 (a measure of non-specific molecular interactions).
The concentration detector employed by the scientists was the Wyatt Technology Optilab rEX, a differential refractive index detector.
Reproducibility of the data is typically better than 1% in molar mass, and the larger of 5% or 2x10^-5 mol/ml/g2 in A2.
