Using Immobilized Protein A or Protein G for Immunoprecipitation
- ImmunoPure® Immobilized Protein A (Pierce product #20333) or ImmunoPure® Immobilized Protein G (Pierce product #20398).
- Primary antibody
- Immunoprecipitation buffer: 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate and 0.02% sodium azide. > NOTE: 50 mM sodium acetate buffer, pH 5.0,. 500 mM NaCl, 0.1% SDS, 1% NP-40 and 0.02% sodium azide may be used as the immunoprecipitation buffer to improve the efficiency of binding with protein G.
- Elution Buffer: Pierce’s ImmunoPure® IgG Elution Buffer (Product No. 21004) or 0.1 M glycine-HCl buffer, pH 2.5.
- SDS-PAGE sample buffer (pH 6.8) containing 2% SDS, 62.5 mM Tris Base, 10% glycerol, 2-5% 2-mercaptoethanol, or use Pierce Product No. 39000.
NOTE: Perform the following experiment in a microcentrifuge tube.
- Solubilize antigen in 50 µl of immunoprecipitation buffer and add a molar excess of primary antibody to the protein solution containing the antigen of interest. Adjust the volume of the sample to 0.2 ml with immunoprecipitation buffer. Typically, 2-5µg of purified antibody will be sufficient for this volume of precipitation mix.
- Incubate the sample overnight at 4°C.
- Add appropriate amount of immobilized protein A or G to the antigen-antibody complex (~ 50 µl of gel per 5 µg of antibody).
- Incubate the sample with gentle mixing for 2 hours at room temperature.
Wash the immobilized protein A or G-bound complexes with 0.5 ml of the immunoprecipitation buffer, followed by centrifugation for 2-3 minutes in a microcentrifuge. Discard the supernatant. Repeat this wash procedure at least 6 times.
- Elute the bound antigen-antibody complex from the immobilized protein A or G by incubation for 5 minutes with 50 µl of the elution buffer. Centrifuge the sample, collect the supernatant, and incubate the gel with another 50 µl of the elution buffer (5 minute incubation). Then, centrifuge the sample and collect the supernatant. Combine the 2 x 50 µl supernatant samples.
- Immediately adjust the protein sample to a physiological pH by addition of a suitable, more concentrated buffer such as 1.0 M Tris, pH 7.5 (10 µl of this buffer to 100 µl of the supernatant should be sufficient).
- Desalt the eluted fraction.
- The sample is ready for characterization.
If the antigen is to be characterized by SDS-PAGE, Steps 6-9 may be eliminated. In this situation, follow the above procedure to Step 5. Upon completing Step 5, wash the gel once with 0.5 ml distilled water. Centrifuge the immobilized protein A or G for 2-3 minutes and discard the supernatant. Then incubate the complex-bound immobilized protein A or G for 5 minutes at 95°C with 25µl of the SDS-PAGE sample buffer. Centrifuge the sample and collect the supernatant. Repeat, pooling supernatant for a total of 50µl. The sample is now ready