Protocols | Immunoprecipitation
Immunoprecipitation
This immunoprecipitation protocol is provided as a guide only. Reagents and final procedures will need to be optimized for species, tissue type and application combination.
Reagents Required
Cell Preparation
- Tris Buffered Saline. Use a 10x TBS, pH 7.5 (1.0M Tris HCl, 1.5M NaCl). Dilute appropriate volume to 1x with de-ionized water. Store at room temperature up to one month.
- Lysis Buffer. TBS containing 1.0% of an appropriate detergent, 1mg.ml bovine serum albumin BSA, and an appropriate proteinase inhibitor.
- Dilution Buffer. Same as lysis buffer without proteinase inhibitor.
- Agarose conjugates for lysate pre-treatment. Pre-absorb lysates to remove non-specific binding to primary and secondary antibodies. Use agarose normal IgG from the same species as the primary antibody and the host secondary antibody. Prepare washed slurry at 1:1 using dilution buffer.
Primary Antibody
- Control for primary antibody. For polyclonal antiserum, use non-immune serum from the same species. For monoclonal antibodies, use the same isotype and purity.
- Agarose conjugates for Immunoprecipitation. Use agarose secondary antibody conjugate against the same species as the primary antibody. Prepare washed slurry at 1:1 using dilution buffer.
- Tris Buffer. Prepare 0.05M Tris buffer, pH 6.8.
- 2x SDS-PAGE Sample Buffer
- 2-Mercaptoethanol
Procedure
- Prepare lysate by incubating 5 x 107 cells in lysis buffer for 30-60 minutes on ice.
- Vortex lysate and centrifuge for 10 minutes at 250 x g to remove nuclei. Retain supernate.
- Clarify supernatant by centrifugation for 30 minutes at 100,000 x g or microcentrifuge for 30 minutes at 10,000 x g.
- Pre-treat lysate to remove nonspecific protein binding by adding agarose conjugate. Use 10µl of control agarose per 200 µl lysate. Shake for 1 hour at 4ºC. Centrifuge at 200 x g. Save supernatant.
- Add 200 µl of pretreated lysate containing antigen to each of two microfuge tubes. Bring volume to 1 ml with dilution buffer.
- Add primary antibody to one tube. For polyclonal antiserum or ascites fluid use 0.5-5 µl. For tissue culture supernatant, use 10-100 µl. To the second tube, add an equivalent volume of control for primary antibody. Incubate on ice for one hour.
- For immunoprecipitation add 50µl of agarose conjugate per tube. Mix with gentle shaking for 1 hour at 4ºC.
- Centrifuge tube 1 minute at 200 x g or micro-centrifuge for 5 seconds. Carefully remove the supernatant with a pipette. Gently resuspend pellet in 1 ml dilution buffer. Repeat wash. Follow with a wash in TBS and then a final wash in 0.5 M Tris, pH 6.8.
- Centrifuge again as above. Add 20-50 µl of sample buffer. Mix and heat for 5 minutes at 100 ºC. Micro-centrifuge briefly and apply supernatant directly to non-reducing SDS-PAGE. If reducing conditions are desired, transfer the supernate to a new tube and add 5% 2-mercaptoethanol. Mix and heat as above.
- Electrophorese protein mixture. Stain gel or immunoblot (using our Western Blot Protocol) to visualize. Bands present will include polypeptides of antigen and antibodies used.
How to choose the correct beads
| Species immunoglobulin isotype | Protein A | Protein G |
|---|---|---|
| Human IgG1 | Strong Binding | Strong Binding |
| Human IgG2 | Strong Binding | Strong Binding |
| Human IgG3 | No Binding | Strong Binding |
| Human IgG4 | Strong Binding | Strong Binding |
| Human IgM | Use anti human IgM | |
| Human IgE | No Binding | Weak Binding |
| Human IgA | No Binding | Weak Binding |
| Mouse IgG1 | Weak Binding | Strong Binding |
| Mouse IgG2a | Strong Binding | Strong Binding |
| Mouse IgG2b | Medium Binding | Medium Binding |
| Mouse IgG3 | Weak Binding | Weak Binding |
| Mouse IgM | Use anti Mouse IgM | |
| Rat IgG | No Binding | Weak Binding |
| Rat IgG2a | No Binding | Strong Binding |
| Rat IgG2b | No Binding | Medium Binding |
| Rat IgG2c | Weak Binding | Medium Binding |
| Chicken all isotypes | No Binding | No Binding |
| Cow all isoptypes | Medium Binding | Strong Binding |
| Goat all isotypes | No Binding | Medium Binding |
| Guinea Pig all isotypes | Strong Binding | Medium Binding |
| Hamster all isotypes | Weak Binding | Medium Binding |
| Horse all isotypes | Weak Binding | Strong Binding |
| Pig all isotypes | Weak Binding | Medium Binding |
| Rabbit all isotypes | Strong Binding | Medium Binding |
| Sheep all isotypes | No binding | Strong Binding |