- Gel Permeation and Size Exclusion Chromatography;
- High Five (Stephanie Plum, Book 5).
- Comparison size-exclusion chromatography (SEC) / Analytical Ultracentrifugation (AUC).
Figure 2 - Hydrodynamic diameter z -average, nm, in blue and light scattering intensity kilo counts per second, in green versus elution volume for BSA, injected onto a Superdex column. Figure 2 shows the z -average size DLS and the light scattering intensity trace. The monomer peak eluted between This is very close to the expected size for this molecule. On the basis of this size, the computed molecular weight prediction was 72 kDa, slightly larger than the 67 kDa expected. Nevertheless, this estimate clearly confirms that this material is the BSA monomer, rather than any other oligomer.
Gel Permeation Chromatography (GPC Analysis)
In the analysis of the elution profile, the majority by intensity is found to be a monomer. Results can also be interpreted in terms of size distribution to give an indication of the relative composition of the original sample. Figure 3 shows the size distribution of the BSA sample. The majority of the protein Figure 4 - Hydrodynamic diameter z -average, nm, in blue and light scattering intensity kilo counts per second, in green versus elution volume for IgG4, injected onto a Superdex column.
How to Estimate the MW using a Gel Filtration Chromatogram
An antibody IgG4 of expected molecular weight around kDa was prepared in 0. Figure 5 - Hydrodynamic diameter z -average, nm, in blue and light scattering intensity kilo counts per second, in green versus elution volume for carbonic anhydrase. The main monomer peak eluted between This is close to the expected size for this molecule.
A prediction of the molecular weight from the measured size leads to kDa, rather more than the nominal kDa expected. The higher size is due to the Y-shape of the IgG4 molecule—antibodies do not give an ideal fit with the globular protein family model. A typical compact globular protein of kDa would have a size of 10 nm. Nevertheless, the estimate of kDa clearly confirms the presence of the monomer.
Protein Aggregate Analysis
The single peak, eluted between This simple size measurement confirmed the main elution peak as that of the carbonic anhydrase monomer. The latest generation of DLS systems, specifically the Zetasizer Nano, allow the coupling of dynamic light scattering and size exclusion chromatography in the form of absolute SEC.
A powerful, easy-to-use protein characterization tool, ASEC has the advantage of providing a rapid and direct measurement of protein size without the need for costly and time-consuming column calibration.
In addition, since size versus mass relationships for globular proteins are well known, DLS measurements of the hydrodynamic radius can also be used to calculate molecular weight. The same technique is applicable to other types of chromatographic measurement, such as affinity or ion exchange chromatography , thereby enhancing the experimental return.
About dynamic light scattering Dynamic light scattering is a powerful technique for determining structural characteristics such as molecular weight, size, melting point, and charge zeta potential of particles in solution. One of its great advantages is its ability to measure a large population of particles in a very short time period, with no manipulation of the dispersing medium.
The Zetasizer Nano Figure 6 is a benchtop instrument that includes both the hardware and software for combined dynamic, static, and electrophoretic light scattering measurements. The system was designed specifically to meet the low concentration and sample volume requirements typically associated with pharmaceutical and biomolecular applications, along with the high concentration requirements for colloidal applications.
With a size range of 0. It consists of three unique …. A gel permeation chromatograph GPC is used to separate the components of a sample by size. GPC systems are widely used to analyze polymers and … Learn More. Dynamic light scattering particle size analyzers are used to measure the size of very small particles 0. This class of … Learn More.
Ion chromatography IC systems separate charged particles from a liquid and measure their concentration. IC systems can analyze particles such as … Learn More. Keep up with our latest articles, news and events. Plus, get special offers and more delivered to your inbox.
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- Kinetic Binding Parameters Derived from Size-Exclusion Chromatography?
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- Gel Permeation and Size Exclusion Chromatography;
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