Antibody Purification using Blotted Antigen¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Antibody Purification using Blotted Antigen
- Load antigen onto a preparative (single well) SDS PAGE gel.
- Electro-blot onto Nitrocellulose. (Transfer for 1.5 hours instead of the usual 1 hour).
- Ponceau S stain the blot for 1min. Cut out portion of Blot that contains the Antigen. Wash with copious amounts of distilled water or TBST until the Ponceau S is gone. Keep strip of Antigen in a 15ml conical tube. Block with 5% Milk in TBST for at least 20 min. Rinse 2X (10ml) TBST.
- Add crude serum with antibody. (5-10 ml.). Incubate 4hr to overnight.
- Pour off the unbound fraction and save serum.
- Wash the blot 3X with 10ml TBST.
- Elute bound antibodies with glycine buffer, pH 2.7 and mix/vortex for 5- 10 min. Collect into a new tube and add 1/10 the volume of 2 M Tris, pH 8.0 to neutralize the antibody which will prevent denaturation of antibodies, which can result from low pH. Check pH with pH paper. Measure the concentration of the protein A280. Use the neutralized glycine buffer as blank. (Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)) Use a quartz cuvette not plastic.
For example if eluted with 10ml glycine then add 1ml 2M Tris, pH 8.0. Elute at least 3X.
- Dialyze the antibody in 1XPBS. Lyophilize the Antibody and resuspend in 50% glycerol. Alternatively the antibody may be stored in the neutralized elution buffer.
This method is based, with permission, on an original protocol available here.