By Gebhard von Jagow, Arnold Revzin

A functional advisor to Membrane Protein Purification is written specifically for researchers who've a few familarity with separation of water-soluble proteins, yet who is probably not conscious of the pitfalls they face with membrane proteins. This consultant offers ideas in a concise shape, emphasizing the points special to membrane proteins. The ebook explains the foundations of the equipment, allowing researchers and scholars new to this region to evolve those innovations to their specific wishes. the second one quantity within the sequence, this e-book is a necessary handbook 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 courses. every one quantity specializes in a subject matter of critical curiosity to scientists and scholars in biomedical and organic learn. Introductory chapters are by way of transparent, step by step protocols that current rules and perform. those concise manuals are designed for optimum realizing of tools in addition to for useful benchtop use.

  • Provides normal directions and techniques for isolation of membrane proteins
  • Describes certain functional methods which were the widest functions, and lowest really good apparatus needs
  • Gives particular emphasis to new local and denaturing electrophoresis techniques
  • Explains differences of thoughts used for water-soluble proteins

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Additional resources for A Practical Guide to Membrane Protein Purification, Volume 2 (Separation, Detection, and Characterization of Biological Macromolecules)

Example text

CHAPTER 2 Chromatographic Techniques and Basic Operations 33 Delipidation can be avoided if the bound protein is eluted directly after binding to the column, without a washing step. Exchange of detergents works similarly. Detergent bound to membrane proteins usually is replaced by other detergents present in washing and elution buffers. There is no problem with those membrane proteins that are stable on delipidation; however, many membrane proteins will irreversibly denature when washed at low detergent concentration (slightly above the CMC).

Protein Adsorption and Elution Increasing ionic strength reduces the degree of ionization both of the ion exchanger and of the protein; a lower effective binding affinity results. The effect varies with the counterion used. High-affinity counterions may be used with tightly binding proteins. High affinity Anion exchanger Cation exchanger I~ > N0 ~ 3 Low affinity > H P ( V > CI" > H C C V > C H C O C T > OH~ > F " 2 Cs + 3 > Rb > K + + > NH 4 + > Na + > H + > Li + The affinity of proteins for the column phase can also be regulated by adjusting the pH.

5). 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.

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