A Practical Guide to Membrane Protein Purification, Volume 2 by Gebhard von Jagow, Arnold Revzin

By Gebhard von Jagow, Arnold Revzin

A sensible advisor to Membrane Protein Purification is written specifically 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 elements specified to membrane proteins. The booklet explains the foundations of the tools, allowing researchers and scholars new to this sector to conform those strategies to their specific wishes. the second one quantity within the sequence, this publication is a vital 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 important curiosity to scientists and scholars in biomedical and organic examine. Introductory chapters are by means of transparent, step by step protocols that current ideas and perform. those concise manuals are designed for optimum realizing of equipment in addition to for useful benchtop use.

Key Features
* offers common directions and methods for isolation of membrane proteins
* Describes special useful techniques which have been the widest purposes, and lowest really expert apparatus needs
* offers specific emphasis to new local and denaturing electrophoresis techniques
* Explains changes of recommendations used for water-soluble proteins

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Extra info for A Practical Guide to Membrane Protein Purification, Volume 2

Example text

Chloride is usually used as counterion. Buffers carrying carboxyl groups are also useful, since they have only small interactions with the solid support material. 2), which should not interfere with the cation-exchange process. 3. Initial Steps with Membrane Proteins a. Determining pH- and Detergent-Dependent Stability It is essential to determine the pH- and detergent-dependent solubility and stability of the protein of interest in the supernatant after ultracentrifugation. Knowing the pH stability range is essential for retaining the native state of membrane proteins, but it has little influence on the choice of using either the cation- or the anion-exchange mode.

These substances absorb water, but even small membrane proteins will not enter the pores because of bound detergent. 32 Hermann Schagger The approach is simple and fast. It has the disadvantage that removal of the gel by centrifugation or filtration is required; also, protein is partially lost between the residual gel particles. 3. Dialysis against Inert Dry Media An alternative technique involves placing the protein sample in a dialysis bag and then (a) covering the bag with dry Sephadex G-100 and incubating until sufficient buffer has migrated out of the bag to leave the desired sample volume or (b) placing the bag into a solution of at least 20% PEG (usually PEG 20,000) and dialyzing until adequate volume reduction has occurred.

30 Hermann Schagger Notes: The degree of ammonium sulfate saturation influences the viscosity of the membrane protein layer. Brittle layers at high saturation are easier to handle. Compared with the Triton X-114 phase separation approach described above, the Triton X-100/ammonium sulfate method works at low temperature and uses a less aggressive detergent. No protein denaturation was observed. The phase separation properties of several neutral and ionic detergents as a function of salt concentration, and their use for fractionation of membrane proteins, are described by Parish et al (1986) and Fricke (1993).

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