Knowledge
by
experience

Affinity purification of antibodies - low amount of antigen available

Needed:

  • Serum or total IgY fraction containing antigen specific antibodies

  • Gel electrophoresis and Western Blot equipment

  • Nitrocellulose or PVDF membrane

Short protocol:
  • 1. Run a sample containing your antigen (purified protein or cell extract) on an SDS-PAGE. Note: If antigen solution is very pure it can be directly spotted on the membrane, dried and than continue from point 5.

  • 2. Make a Western Blot transfer.

  • 3. Stain the membrane with Ponceau S.

  • 4. Locate the band corresponding to your antigen, cut out the band and mark the protein side.
  • 5. Destain and block the strip with 3-5% dry milk in PBS for 1 hour in RT.

  • 6. Wash (by shaking) the strip 3-5 times in PBS.

  • 7. Incubate the strip with serum or IgY sample over night in the cold or 2 hours in RT. Important note: It is very important not to use too low amounts of serum or total IgY sample, since they might contain too little antibodies to bind to the antigen.

    Theoretical calculation: 1 mg of the antigen (protein) can theoretically bind around 0.5-2 mg of the specific antibodies. Specific antibodies are from 1-10 % of the total antibody pool. If we take the lowest 1 % of specific antibodies in our sample, 1 ml will contain around 0.01 mg of specific antibodies. If less than 0.5 mg of the antigen is present on the membrane, it should be incubated with at least 10-15 ml of serum or IgY sample.

  • 8. Remove the serum and wash the strip with PBST (1x) and PBS (3x). Measure A280, to be sure it is below 0.1 at the end of the wash.

  • 9. Elute antibodies applying 100 mM glycine pH 2.8-2.5, for 10-30 minutes.

  • 10. Immediately after elution is completed neutralise the eluate with 0.05 ml 100 mM Tris pH 8/1 ml.

  • 11. Wash the strip extensively in PBS, store it afterwards at -20°C.

  • 12. Concentration of the eluted antibody might be difficult to estimate, depending upon the elution volume. To check antibody activity run Western Blot or ELISA. If volume allows measure A280 of a whole sample and calculate antibody concentration. Antibody solution at concentration of 1 mg/ml gives absorbance of 1.36 for IgG and 1.4 for IgY at 280 nm.

  • 13. Note: Elution conditions and time have to be adjusted for each specific antibody.

Last changed: 2009-04-02 by WebMaster