Affinity purification of antibodies - small amount of protein 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.

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