By Gebhard von Jagow, Arnold Revzin
A sensible advisor to Membrane Protein Purification is written in particular for researchers who've a few familarity with separation of water-soluble proteins, yet who will not be conscious of the pitfalls they face with membrane proteins. This consultant offers recommendations in a concise shape, emphasizing the points distinctive to membrane proteins. The e-book explains the rules of the tools, allowing researchers and scholars new to this region to evolve those ideas to their specific wishes. the second one quantity within the sequence, this booklet is an important guide for investigations of constitution and serve as of local membrane proteins, in addition to for purification of those proteins for immunization and protein sequencing.
Separation, Detection, and Characterization of organic Macromolecules is a brand new sequence of laboratory publications. each one quantity makes a speciality of a subject of valuable curiosity to scientists and scholars in biomedical and organic examine. Introductory chapters are through transparent, step by step protocols that current ideas and perform. those concise manuals are designed for optimum figuring out of tools in addition to for useful benchtop use.
* presents normal instructions and techniques for isolation of membrane proteins
* Describes distinct sensible techniques which were the widest functions, and lowest really expert gear needs
* offers precise emphasis to new local and denaturing electrophoresis techniques
* Explains variations of ideas used for water-soluble proteins
Read or Download A Practical Guide to Membrane Protein Purification, Volume 2 PDF
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Extra info for A Practical Guide to Membrane Protein Purification, Volume 2
Here presentation of the basic separation techniques is kept short. More elaborate descriptions of the methods, and applications to isolation of water-soluble proteins, can be found elsewhere (Deutscher, 1990; Harris and Angal, 1992; Janson and Ryden, 1989). A. Hydroxylapatite C h r o m a t o g r a p h y Crystalline hydroxylapatite which allows a high flow rate through columns can be prepared easily as described in Chapter 5, Section II. Various commercial preparations of hydroxylapatite, usually displaying lower flow rates, are available from a variety of suppliers.
However, at the low detergent/ lipid ratios that optimize retention of protein-bound lipid ( 2 - 3 molecules of detergent per molecule of lipid; saturated detergent micelles), membrane proteins often become insoluble. With membrane proteins that are less sensitive to detergent levels, column chromatography is preferred, since greater purity and higher yields are obtainable. B . Ion-Exchange C h r o m a t o g r a p h y Ion-exchange chromatography is often the method of choice in isolation of water-soluble as well as membrane proteins, for a number of reasons, (i) Chromatography media having considerably different binding properties are available at relatively low cost, (ii) High flow rates can often be attained, which allow for large-scale preparations, (iii) The mode of interaction of proteins with the chromatography medium is well defined, and standard binding/ elution conditions can be used, (iv) Any neutral detergent capable of solubilizing membrane proteins can be used for purification of membrane proteins by chromatography.
A 5-fold enrichment was obtained with mitochondria. a. If peripheral membrane proteins are of interest, extract all water-soluble and peripheral membrane proteins from the membranes with Triton X-100 using a preextraction procedure as described above. If integral membrane proteins are the focus, solubilize all proteins including integral membrane proteins at higher Triton X-100 concentrations. b. Add cold saturated ammonium sulfate, pH 7 (preparation is described in Chapter 6), uniformly with stirring.