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Related Experiment Videos

Purification of the basic protein avidin using Gradiflow technology.

Deborah L Rothemund1, Theresa M Thomas, Dennis B Rylatt

  • 1Gradipore Ltd, 22 Rodborough Road, P.O. Box 6126, 2086, Frenchs Forest, NSW, Australia. drothemund@gradipore.com

Protein Expression and Purification
|October 3, 2002
PubMed
Summary

Researchers purified avidin, a basic protein, from chicken egg white using a two-step Gradiflow electrophoresis method. This efficient process achieved good recovery with minimal contamination from other egg white proteins.

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Area of Science:

  • Biochemistry
  • Proteomics
  • Analytical Chemistry

Background:

  • Avidin is a basic protein found in chicken egg white, known for its high affinity to biotin.
  • Purification of avidin is crucial for various biochemical and biotechnological applications.
  • Traditional purification methods can be time-consuming and may result in lower yields.

Purpose of the Study:

  • To develop an efficient and rapid method for purifying avidin from chicken egg white.
  • To utilize preparative electrophoresis for protein separation based on charge and size.
  • To assess the recovery and purity of avidin obtained through the proposed method.

Main Methods:

  • Employed the Gradiflow instrument for preparative electrophoresis.
  • Utilized a sequential two-step charge-based separation protocol.

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  • First step at pH 9.0 to separate positively charged proteins (avidin, lysozyme) from others.
  • Second step at pH 10.2 to separate negatively charged avidin from positively charged lysozyme.

    • Main Results:

    • The two-step protocol successfully purified avidin within 4.5 hours.
    • Achieved avidin recoveries ranging from 60-65% from a single egg white.
    • Minimal lysozyme activity was detected in the purified avidin fractions, indicating high purity.

    Conclusions:

    • The Gradiflow preparative electrophoresis is an effective tool for purifying basic proteins like avidin.
    • The developed sequential charge-based separation protocol is rapid and efficient.
    • This method offers a viable alternative for obtaining high-purity avidin for research and applications.