Immunoprecipitation Protocol
This protocol outlines the reagents, buffer and step-by-step procedure to be used with immunoprecipitation experimentation. To be used with immunoblotting and western blotting research methods.
Materials
- Immunoprecipitation (IP) lysis buffer
- Protease Inhibitors (Calbiochem Cat#539131)
- Primary Antibodies made in Rabbit
- Normal IgG, negative control (Rabbit IgG- Bethyl Cat. No. P120-101)
- Protein A Sepharose Beads (Amersham Cat# 17-0780-01)
- Cell Lysate
- Sample Buffer
Preparation
IP lysis Buffer
- 12.5 ml 1M NaCl (250mM)
- 2.5 ml 1M Tris (50mM)
- 500ul 0.5M EDTA (5mM)
- 2.5ml 10% NP-40
- 32 ml dH20
Protein A Beads
- Resuspend 400 mg of Protein A beads in 10 ml of distilled H2O
- Mix well to resuspend
- Spin at 250 rpm for 5 minutes.
- Wash 3X in 10 ml IP Lysis buffer.
- Resuspend to 10 ml with IP lysis buffer for a 20% solution. Use 100 mcl per IP reaction.
4X Sample Buffer
- Glycerol 4.0 g
- Tris Base 0.68 g
- Tris HCL 0.67 g
- LDS 0.80 g
- EDTA 6 mg
- Brilliant Blue G250 2.5 mg
- Phenol red 2.5 mg
- Adjust volume to 10 ml with ultra pure water.
Store at 4 °C.
4X sample buffer is available from Invitrogen (Cat# NP0007)
1X Sample Buffer
- 4X sample buffer 150 mcl
- 1M DTT 60 mcl
- Distilled water 390 mcl
Make fresh for each use.
Procedure
- Prepare cell lysate according to protocol. Place 500 mcl of the prepared cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
- To this tube add 2 to 10 mcg of the primary antibody (If using neat sera or an IgG fraction such as Protein-A purified antibody, larger amounts are likely to be required. For best results, optimal amounts of antibody should be empirically defined.)
- To a negative control reaction, add an equivalent amount of normal rabbit IgG.
- Add 100 mcl of a 20% Protein A suspension.(Amersham Biosciences, Cat# 17-0780-01) to the mixture of antibody and cell lysate. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 °C.
- Centrifuge (200 x g; 5 minutes) to pellet the complex.
- Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
- Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
- After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 °C for 5 minutes.
- Continue with electrophoresis and immunoblotting as described under western blotting protocol. Load 8 to 16 mcl (20 to 40% of the IP reaction) to a polyacrylamide gel.
Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.
