Protein Blotting from Polyacrylamide Gels on Glass Microfiber Sheets: Acid Hydrolysis and Gas-Phase Sequencing of Glass-Fiber Immobilized Proteins

  • Joel Vandekerckhove
  • Guy Bauw
  • Magda Puype
  • Jozef van Damme
  • Marc van Montagu

Abstract

The transfer onto immobilizing membranes of proteins which are separated on polyacrylamide gels (popularly referred to as the Western blot) has become an important tool in protein chemistry (for a comprehensive review see [1]). Originally, transferred proteins were covalently bound onto the membrane [2, 3], but this method showed low coupling yields and was therefore replaced by the introduction of microporous nitrocellulose membranes to which proteins could be bound by noncovalent interactions [4]. This principle was later improved by using a nylon-based, positively charged membrane [5]. Although proteins immobilized in this way could be easily assayed for their antigenicity or their enzymatic or lectin-binding activity [1], the membranes used for these analyses were sensitive to the chemical treatments necessary for acid hydrolysis or for the Edman-de-gradation-based chemistry. In order to combine the simple, fast, inexpensive, high-resolution purification procedure of polyacrylamide gel electrophoresis with the recently developed technique of protein gas-phase sequencing [6], it was necessary to introduce a support which is both able to bind every kind of protein eluting from a gel, and resistant against chemicals and solvents used in Edman chemistry.

Keywords

HPLC Glycine Phenyl Immobilization Methionine 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1986

Authors and Affiliations

  • Joel Vandekerckhove
    • 1
  • Guy Bauw
    • 1
  • Magda Puype
    • 1
  • Jozef van Damme
    • 2
  • Marc van Montagu
    • 1
  1. 1.Laboratory of GeneticsState University GentGentBelgium
  2. 2.N.V. Plant Genetic SystemsGentBelgium

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