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Benefits of tetra detection for SEC analysis of membrane proteins revealed!

10 October 2014 No Comment

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Membrane protein

As a Product Technical Specialist at Malvern Instruments a great part of my job is talking to customers and finding out how they are utilizing our instruments to perform new and exciting research. My role focuses on the use of Size Exclusion Chromatography (SEC) to analyze biopolymers and proteins. Following on from my recent blog post about Hyaluronic Acid I want to highlight protein research and more specifically membrane proteins.

Part of my role is to keep up to date with the latest information being published which involves me reading a significant number of research papers. As part of this I came across a fantastic paper by Dr. Larry Miercke and his colleagues at University of California, San Francisco titled “Tetra Detector Analysis of Membrane Proteins”. This paper covers one of the fundamental roles of scientific literature, the dissemination of methodologies for other researchers.

Before talking more about this paper I think it is important for me to discuss why I am so interested in these material types. Membrane proteins play a crucial role in biology and further understanding their structure-function relationships can make a significant contribution to structural biology and biopharmaceutical development. As the name suggests, membrane proteins are typically associated with the lipid cell membrane and can either cross the membrane entirely or be associated with part of it. When these proteins are purified from the cell they are very fragile and need to be stabilized by the substitution of the lipid with detergent, to form a membrane Protein Detergent Complex (PDC).

It is known that PDC’s can have unfavorable properties with regard to their structural analysis, which may manifest as their inability to crystallize for NMR or X-ray crystallography. In order to improve their ability to crystallize it is important to optimize the detergent associated with the protein, either by changing the detergent used or by changing the purification process. However, it is difficult to measure the stoichiometry of a PDC as the amount of detergent associated with a protein following the purification techniques can depend both on the protein and the detergent.

SEC is crucial for the production and purification of PDC’s. Typically this technique uses a single concentration detector to record the elution volume of a desired material to allow the user to fractionate the product. The combination of SEC with light scattering detectors gives the user significantly more information. As well as allowing the absolute molecular weight of a sample to be determined it also provides the stoichiometry of the sample, and a measure of how much protein and detergent are associated together. This information can be extremely important and is used to help improve the likelihood of a sample to form crystals.

The paper produced by Dr. Miercke covers the procedural steps necessary to measure the absolute molecular weight and stoichiometry of a PDC sample using the Tetra Detection Array (TDA). In addition to covering the theory behind the system, the steps necessary to obtain material constants such as the refractive index increment of the detergent, are described.

I would highly recommend that anyone interested in membrane proteins reads this paper – click here to access it.  If you are interested in measuring proteins using SEC, you should also watch our recent webinar – Powerful protein SEC made simple! (click here to view) – reveals how Malvern’s protein SEC measurement capabilities within the Viscotek range have improved in the last year, and also introduces the latest release of OmniSEC which includes a new Bio interface, bringing protein-specific information to the forefront and simplifying protein analyses.

Further Reading:

Application Note: Analysis of membrane protein by multi-detector SEC