Coupling of Antibodies to Protein A Beads

In order to immunoprecipate antigens, the revelant antibody is preferably coupled to the solid support (protein A-sepharose beads), but alternatives are mentioned in Note 5.

1. Wash the protein A-sepharose beads with PBS, and then add the monoclonal antibody (MAb) (see Note 6) to 2 mg/mL of packed protein A-sepharose beads in PBS for 2 h at 4°C. Fifty millimeters of ascites or 1 mL of polyclonal antiserum or tissue-culture supernatants/mL of packed beads can also be used.

2. Wash in 0.1 M borate buffer, pH 8.2, three times, and then once with 0.2 M triethylamine, pH 8.2.

3. Add 5 vol of 0.2 M triethylamine, pH 8.2, containing 20 mM dimethyl pimelimidate for 45 min to crosslink the protein to the beads.

4. Spin down the beads, and add 20 mM ethanolamine, pH 8.2, to stop the reaction. After 5 min, wash three times with 0.1 M borate buffer, and finally resuspend the coupled beads in RIPA or NP40 lysis buffer (Note 7).

Fig. 1. Schematic diagram of the relationship of the protocols described in this chapter.

Was this article helpful?

0 0

Post a comment