Technical information > Antibody purification - small amount of protein

Material

• Serum or total IgY fraction containing antigen specific antibodies
• Equipment for protein gel electrophoresis and Western Blot
• Nitrocellulose or PVDF membrane
   

Short protocol

1. Separate an equivalent of around 800 µg of sample containing your antigen (purified protein or cell extract) on an SDS-PAGE. The sample can be loaded into all lanes made with a 12-15-lane comb, or into a special comb for 2nd dimension gel electrophoresis. Note: If the antigen solution is very pure, it can be spotted directly onto the membrane and dried, and the protocol can then be continued directly from step 5.
2. Transfer the resolved proteins onto the membrane.
3. Stain the membrane with Ponceau S (Figure A).
   
   
   
4. Locate the band corresponding to your antigen, cut out the band and mark the protein side (Figure B).
5. Destain and block the strip with 3-5% dry milk in PBS for 1 h in RT.
6. Wash (by shaking) the strip 3-5 times in PBS.
7. Incubate the strip with serum or IgY sample overnight in the cold or for 2 h at RT.
   Note: It is very important not to use too low amounts of serum or total IgY sample, since they might contain too low levels of the antibody that bind to the antigen.
   Theoretical calculation: 1 mg of the antigen (protein) can theoretically bind around 0.5-2 mg of the specific antibody. The specific antibody constitute from 1-10% of the total antibody pool. If we consider the lowest percentage of specific antibody in our sample (1%), 1 ml will contain around 0.01 mg of the specific antibody. 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 (save it) and wash the strip with PBST (1x) and PBS (3x). Measure A280, to make sure that it is below 0.1 at the end of the wash.
9. Elute the antibodies by applying 100 mM glycine pH 2.8-2.5, for 10-30 min.
10. Immediately after elution is completed, neutralize the eluate with 0.05 ml 100 mM Tris pH 8 per each 1 ml.
11. Wash the strip extensively in PBS, and store it afterwards at -20°C.
12. The concentration of the eluted antibody might be difficult to estimate, depending upon the elution volume. To check the antibody activity, run Western Blot or ELISA. If the volume allows it, measure A280 of a whole sample and calculate antibody concentration. An antibody solution at a concentration of 1 mg/ml gives absorbance of 1.36 for IgG and 1.4 for IgY at 280 nm.