Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatant

Helen M. Cooper1, Yvonne Paterson2

1 Walter and Eliza Hall Institute, Melbourne, Australia, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.14
DOI:  10.1002/0471142727.mb1114s50
Online Posting Date:  May, 2001
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Abstract

This unit describes the isolation of the immunoglobulin G (IgG) fraction (containing antibodies of all specificities) from a complex protein mixture such as antiserum, ascites fluid, or hybridoma supernatant. The basic procedure utilizes saturated ammonium sulfate solution to precipitate the IgG fraction, while an alternate protocol describes fractionation of IgG by chromatography on DEAE‐Affi‐Gel Blue resin.

     
 
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Table of Contents

  • Basic Protocol 1: Precipitation of IgG with Saturated Ammonium Sulfate
  • Alternate Protocol 1: Fractionation of IgG by Chromatography on DEAE–AFFI‐Gel Blue
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Precipitation of IgG with Saturated Ammonium Sulfate

  Materials
  • recipeSaturated ammonium sulfate (SAS) solution (see recipe)
  • recipe33% SAS solution (see recipe)
  • Immunoglobulin‐containing antiserum, ascites, or tissue culture supernatant
  • Additional reagents and equipment for dialysis ( appendix 3C)

Alternate Protocol 1: Fractionation of IgG by Chromatography on DEAE–AFFI‐Gel Blue

  • recipeLoading buffer (see recipe)
  • recipeElution buffer (see recipe)
  • NaN 3, crystalline form
  • DEAE–Affi‐Gel Blue (Bio‐Rad)
  • Additional reagents and equipment for gel‐filtration chromatography (unit 10.9) and SDS‐polyacrylamide gel electrophoresis (unit 10.2)
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Literature Cited

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