10: Protein purification
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Reading & Problems: LNC p. 89-95, p. 112 prob. 15, 16
I. Gel electrophoresis - use of an electric current to move molecules in the solvent phase through a gel matrix. Gels can be made of hydrophilic plastics (e. g. polyacrylamide) or carbohydrates (e. g. agarose - a long-chain polysaccharide purified from marine algae).
A. SDS polyacrylamide gel electrophoresis (SDS-PAGE)- proteins denatured with SDS (sodium dodecyl sulfate, a detergent) and separated by size (smaller runs more rapidly) on polyacrylamide gels. SDS -PAGE Animation
Here are examples of a commassie stained gel from Dr. Gasser's research lab, and an autoradiogram of a gel of radioactively labeled proteins from Dr. Gasser's dissertation research.
B. Isoelectric focusing electrophoresis (IEF) - polyampholytes establish a pH gradient and proteins are separated according to pI.
An example of a polyampholyte that can be used in IEF (parentheses indicate variable numbers and ordering of the groups).
A. Gel filtration - separates by size. Large molecules are excluded from pores in beads and flow through rapidly (the "excluded fraction"), small molecules enter the beads, retarding their flow, and they move through slowly (the "included fraction").
B. Ion exchange - used for proteins just as for amino acids, separates by charge/pI. Columns can be negatively or positively charged and can be eluted with changes in pH or salt concentration or a combination.
C. Reverse phase - separates by hydrophobicity. Hydrophobic groups on beads bind hydrophobic regions of proteins. Columns are eluted by increasing salt concentration or by increasing the concentration of organic solvent (e. g. acetonitrile, methanol, propanol, tetrahydrofuran) in the solution.
Some take home information:
Analytical procedures: performed when you want to determine "what is in a sample". Desirable properties of such a procedure include:
- High resolution (so you can separate molecules with only slightly different properties)
- High sensitivity (so you don't have to use much sample to get the desired information)
- Ability to process a number of samples in parallel (so you can get more information from a single experiment)
Electrophoresis methods are commonly used for analysis because of their high resolution, high sensitivity and the parallel nature of gel methods, but modern column methods can also have sufficient resolution and can often be more easily adapted to automated analysis of samples.
Preparative procedures: performed when you want to obtain a sample of a particular compound purified from a mixture. Desirable properties include:
- High capacity (i. e. ability to process large amounts of material, so that you can obtain sufficient material for further use)
- Ease of utilization of the material once separated (e. g. material from columns is usually in a liquid form that can be used directly, whereas samples separated on a gel must be extracted from the gel matrix).
However, capacity must be balanced with resolution. In some cases you may need high resolution to get a sufficiently pure sample and might be willing to settle for lower capacity if you are able to work with only a small amount of purified material. Column methods are commonly used for preparative procedures, but when resolution is critical, high capacity is not needed, and/or parallel purification is desired preparative electrophoresis methods can be used.
Charles S. Gasser (Department of Molecular & Cellular Biology; UC Davis)