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Antibody Affinity Purification   

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Antibody purification is performed to concentrate and enrich antigen-specific antibodies and lower the background signal during detection by removing any non-specific proteins. Affinity purification makes use of specific binding interactions between molecules, and is very simple and efficient.

Materials and Reagents

  1. PBS
  2. NaCl
  3. KCl
  4. Na2HPO4
  5. KH2PO4
  6. Glycine
  7. HCl
  8. Tris-HCl
  9. BSA
  10. Azide
  11. Affinity resin
  12. Sepharose
  13. 10x PBS (see Recipes)
  14. 1x PBS + 1 M NaCl (see Recipes)
  15. 0.1 M glycine-HCl (pH 2.8) (see Recipes)
  16. 1 M Tris- HCl (pH 8.5) (see Recipes)


  1. Centrifuges
  2. Affinity resin
  3. Spectrometer


  1. Mix serum with affinity resin (protein coupled to sepharose). If using whole serum, dilute 1: 10 with PBS. Incubate overnight with gentle agitation.
  2. Pour resin into column and collect the flow through. This is 1x pre-absorbed immune serum.
  3. Wash the column with 1x PBS (pH 7.5) until the OD280 is zero.
  4. Wash the column with PBS + 1 M NaCl to elute non-specifically absorbed material until the OD280 is zero.
  5. Wash the column with PBS until the OD280 is zero.
  6. Wash the column with 10 volume of 0.1 M glycine-HCl to elute specifically bound material. The acid eluant is neutralized by placing an aliquot (1/10 of fraction volume) of 1 M Tris-HCl (pH 8.5) into the fraction collection tubes. This is done prior to elution with acid. Collect the entire trailing peak-the highest affinity antibodies are released most slowly.
  7. Wash the column with PBS until the OD280 is zero.
  8. Pool the anitbody fractions and dialyze overnight against PBS/azide. After dialysis, check the OD, add BSA to 1%, aliquot and store at -70 °C.


  1. 10x PBS (pH 7.5)
    40 g NaCl
    1 g KCl
    7.2 g Na2HPO4
    1.2 g KH2PO4
    500 ml
  2. 1x PBS + 1 M NaCl
    25 ml 10x PBS
    14.61 g NaCl
    250 ml
  3. 0.1 M glycine-HCl (pH 2.8)
    0.75 g glycine in 100 ml, pH with 1 N HCl, make fresh.
    4. 1 M Tris- HCl (pH 8.5)
    2.4 g Tris in 20 ml


  1. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3): 218-229.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pang, H. (2012). Antibody Affinity Purification. Bio-101: e208. DOI: 10.21769/BioProtoc.208.

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