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)
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.
- 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
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.