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Protein desalting by isoelectric focusing in a segmented immobilized pH gradient | JoVE Visualize

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Protein desalting by isoelectric focusing in a segmented immobilized pH gradient

P G Righetti¹, B Barzaghi, M Faupel

¹Chair of Biochemistry, Faculty of Pharmacy, University of Milan, Italy.

Journal of Biochemical and Biophysical Methods|December 1, 1987

View abstract on PubMed

Summary

This study shows a new protein purification method using Immobiline gel for efficient desalting. The apparatus rapidly removes salts from protein samples, significantly reducing salt content.

Area of Science:

Biochemistry
Separation Science
Biotechnology

Background:

Protein purification often requires efficient desalting.
Existing methods can be time-consuming or inefficient.
A novel apparatus based on segmented Immobiline gel was previously described.

Purpose of the Study:

To demonstrate the efficacy of the Immobiline gel apparatus for protein desalting.
To evaluate the desalting performance with different salt types and buffers.
To assess the impact of buffer composition on desalting efficiency and time.

Main Methods:

Utilizing a segmented Immobiline gel apparatus with orthogonal hydraulic and electric flows.
Focusing impurities in the Immobiline gel while retaining the isoelectric protein in a flow chamber.
Recycling protein samples through the apparatus to remove salt ions.

Main Results:

Efficient desalting of Hemoglobin A samples (50 mM NaCl or ammonium acetate) achieved in 2 hours.
Salt content reduced by over 10,000-fold, to less than 0.005 mM.
Slower desalting (approx. 20 hours) observed with polyprotic buffers due to matrix interactions, necessitating pH control.

Conclusions:

The Immobiline gel apparatus is highly effective for rapid protein desalting.
Desalting efficiency is influenced by buffer composition, with polyprotic buffers requiring longer times and pH management.
The apparatus utilizes isoelectric membranes for effective protein retention during desalting.

Chemical Engineering

Separation technologies

Protein Desalting By Isoelectric Focusing In A Segmented Immobilized Ph Gradient