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RESEARCH DIVISION Laboratory Manual

 


 

Affinity purification of antibodies from crude serum

(Adapted from the method of Wally Langdon and Christine Thien, UWA)

  1. Binding Antigen to Column **perform all steps at 4C.

  1. Depending on application, affigel-10 or affigel-15 (Biorad catalogue #153-6046 and 153-6052) are appropriate as supports for binding of antigen. Affigel-10 is best for binding proteins at near to or below their isoelectric point, while affigel-15 is better for binding proteins near or above their isoelectric point. Thus, when coupling at or close to neutral pH, affigel-10 is better for proteins with pI of 6.5 to 11.0 while affigel-15 is better for proteins with pI less than 6.5. The following conditions refer to affigel-10 coupling at pH 7.5.
  2. Wash 1ml of affigel beads two times in 10ml cold dH20, spin down between washes at 3500rpm for ca. 30". Resuspend beads in 1ml binding buffer (100mM MOPS, pH 7.5)
  3. Dialyse 4mg antigen protein (at about 1mg/ml) against 1 litre 100mM MOPS pH 7.5 overnight at 4C. Retain 10ul of protein for assaying binding.
  4. Add to beads and agitate gently for 4 hours at 4C.
  5. Take 10ul supernatant and run on SDS PAGE gel with sample from 3 above to assess how much protein has bound column. Samples can also be taken during binding to assess rate of binding. Expect to see at least 90% of the protein bound after 2 hours and complete binding after 4 hours. If binding has not gone to completion, adding CaCl2 to 80mM can improve binding.
  6. Add 100ul 1M ethanolamine HCl pH8, agitate gently for another hour at 4C. This will block any free binding sites on the resin.
  7. Transfer gel to econocolumn (Biorad).
  8. Wash column with the following solutions:
    - 10ml 100mM MOPS pH 7.5
    - 10ml PBS
    - 10ml 100mM glycine HCl pH 2.4 / 150mM NaCl
    - 10ml PBS
  9. Store column in PBS/0.2% sodium azide at 4C.
 

Binding antibodies to column

  1. Wash column with the following solutions:
    - 10ml PBS,
    - 10ml 100mM glycine HCl pH 2.4 / 150mM NaCl
    - 10ml PBS
  2. Spin serum (ca 1.5ml) 13000 rpm 10 minutes.
  3. Apply supernatant to the column to which a 27 gauge needle has been fitted to slow rate at which column flows. Collect run-through and apply to column at least twice more. remove the needle.
  4. Wash column with 10ml PBS.
  5. Elute antibody with 100mM glycine HCl pH 2.4 / 150mM NaCl. Collect at least ten 1ml fractions into 200ul 1M Tris HCl pH 8, to immediately neutralise.
  6. Run 20 ul samples of the fractions on SDS PAGE to assay elution of specific antibody. Antibody should be in fractions one to four.
  7. Wash column with the following solutions:
    - 10ml PBS,
    - 10ml 100mM glycine HCl pH 2.4 / 150mM NaCl
    - 10ml PBS
  8. Store column in PBS/0.2% sodium azide at 4C.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998