Immunochemistry Protocols: Immunoprecipitation

Typically, complete immunoprecipitation of radio-labeled antigen from extracts of transfected mammalian cells requires between 1-5 L of polyclonal antiserum, 5-100 L of hybridoma tissue culture medium, or 1-3 L of ascites. If more antibody is used than is necessary, non-specific background will increase. The concentration of immunoglobulins in antiserum is approximately 6-8 mg/mL. Supernatant from cultured hybridoma cells typically contains 10-100 g/mL immunoglobulin, and ascites fluid contains 5-7 mg/mL immunoglobulin.

Step 1:	Divide the preparation of antigen into two equally sized aliquots and place in microfuge tubes. Adjust the volume of each aliquot to 0.5 mL with I.P. Buffer*. To one aliquot, add antibody directed against the target protein. To the other aliquot, add the same volume of a control antibody. Gently rock both aliquots for 1 hour at 4C.
Step 2:	Add Protein-G Sepharose to the antigen-antibody mixture, and incubate for 1 hour at 4C on a rocking platform.
Step 3:	Centrifuge the Protein-G Sepharose antibody-antigen complex at 10,000xG for 20 seconds at 4C in a microfuge. Remove the supernatant by gentle aspiration. Add 1 ml of Wash Buffer** and re-suspend the Sepharose beads.
Step 4:	Incubate the re-suspended beads for 20 minutes at 4C on a rocking platform. This allows time for the I.P. buffer to equilibrate with the fluid trapped between the beads.
Step 5:	Repeat steps 3 and 4 three times. Collect the final washed Protein-G Sepharose antibody-antigen complex by centrifugation at 10,000xG for 20 seconds at 4C in a microfuge. Take care to remove the last traces of the final wash
Step 6:	Add reducing gel-loading buffer and boil for 3 minutes.
Step 7:	Remove the Protein-G Sepharose from the complex by centrifugation at 10,000xG for 20 seconds at room temperature in a microfuge. Transfer the supernatant to a fresh tube and separate the sample by method of electrophoresis.
	*IP Buffer 50 mm Tris HCl (pH 7.5) 150 mM NaCl 0.1% Tween 20 (or 0.1% Nonidet P 40 (NP 40)) 1 mM EDTA (pH 8.0) 0.25% Gelatin 0.02% Sodium Azide	**Wash Buffer 50 mm Tris HCl (pH 7.5) 150 mM NaCl 0.1% Tween 20 (or 0.1% Nonidet P 40 (NP 40)) 1 mM EDTA (pH 8.0)