Immunoprecipitation Procedure

Required Equipment

• Microcentrifuge
• 1.5 ml microcentrifuge tubes
• Rocker platform
• Scintillation counter
• GF/C Whatman filters
• X-ray film or phosphoimager equipment

Solutions and Reagents

• Monoclonal or polyclonal antibody
• PBSTDS: Mix 100 ml of PBS with 10 ml of 100% TRITON^® X-100 (Cat. No. 648462), 5 g of sodium deoxycholate (Cat. No. 264101 or 264103), and 1 g of sodium dodecyl sulfate (Cat. No. 428015 or 428016); include a cocktail of protease inhibitors - 0.5 mg/ml of leupeptin (Cat. No. 108975), 1 mM EDTA (Cat. No. 34103), 1 mg/ml of pepstatin A (Cat. No. 516481), and 0.2 mM PMSF (Cat. No. 52332); add distilled water to 1L
• One of the following: see the table that follows for a list of Protein A/G affinities for IgG from various species

• Goat anti-rat IgM/Protein G Plus-agarose: for rat IgM monoclonal antibodies:

         - Add 1.5 ml of Protein G Plus-agarose (Cat. No. IP04) to 100 mg of goat anti-rat IgM and rotate for 2-4 hours at room temperature or 6-18 hours at 4^oC
         - Collect the pellet by centrifugation at 1500 rpm
         - Wash with 3 changes of PBS
         - Add PBS to a final volume of 1.5 ml and store at 4^oC; addition of NaN₃ is recommended for storage

• Similarly, other second-step reagents can be prepared as necessary for other IgM antibodies
• Immunoglobulin from normal animals
• 10% TCA (trichloroacetic acid)
• Scintillation fluid

Procedure

NOTE: this procedure is written for the use of agarose beads as the precipitating reagent, however, the procedure can be modified for the use of PANSORBIN^® cells if preferred (please see specific instructions for use of these reagents under the individual catalog numbers)

1. Pre-clear the lysate or conditioned media before using it for immunoprecipitation by adding 1 mg of normal IgG from the appropriate species to the sample together with 20 ml of the agarose conjugate of choice; this will remove proteins that precipitate non-specifically and would thus contaminate the immunoprecipitate
2. Incubate for 1 hour at 4^oC; centrifuge the sample at 1500 rpm to pellet the agarose; carefully collect and save the supernatant fluid and use for specific immunoprecipitation
3. For radiolabeled samples:
   A. Determine the TCA precipitable counts in the radiolabeled sample by spotting 5 ml of pre-cleared sample onto two GF/C Whatman filters; allow 1 filter to air dry (total cpm in the sample) and place the second filter in a small beaker of ice cold 10% TCA for 5 minutes
   B. Transfer the filter from the TCA into a beaker containing 95% ethanol for 5 minutes; remove the filter and air dry
   C. Place filters into 3 ml of scintillation fluid and count the sample
   D. Determine the percent precipitable counts by dividing the cpm obtained after TCA precipitation by the total cpm in the non-TCA precipitated sample; this value should be >90%
4. For unlabeled samples determine the total protein in the sample
5. To a microcentrifuge tube add cell lysate (20-30 x 10⁶ TCA precipitable cpm for radiolabeled samples or 10 mg of protein for unlabeled samples), 1 mg of purified primary antibody (polyclonal or monoclonal), 15 ml of packed agarose beads of choice (i.e. Protein A-agarose, Protein G-agarose, etc.); cap the tubes, place them in a bag, and incubate at 4^oC on a rocker platform for 2-24 hours; NOTE: optimal conditions for immunoprecipitation must be determined empirically for a given antibody and sample
6. Collect the immunoprecipiate by centrifugation in a microcentrifuge at 2500 rpm for 15 minutes, 4^oC
7. Carefully aspirate and appropriately discard the radioactive supernatant (for radiolabeled samples); wash the pellet with 4 changes of PBSTDS, 1 ml per wash, repeating the centrifugation step with each wash
8. Following the final wash aspirate and discard the supernatant and resuspend the pellet in 40-50 ml of SDS-PAGE sample loading buffer
9. Analyze samples by SDS-PAGE
   A. Visualize radiolabeled samples by exposure to X-ray film or using a phosphoimager
   B. Visualize unlabeled samples by western blotting

Notes

• This procedure can be used for cells labeled with other radioactive amino acids (e.g. ¹⁴C or ³H) or with ³²P-orthophosphate; cell labeling must be carried out in a medium lacking the correct amino acid or in phosphate-free medium, as appropriate
• For western blotting use an antibody from a different species than the one used to immunoprecipitate in order to avoid detecting the precipitating antibody with the secondary antibody used for detection; alternatively, use a primary antibody that is directly conjugated to a detector system or to biotin (to be used with a streptavidin detector system)
• It is useful to remove approximately 5 mm from the end of the disposable pipette tips before pipetting concentrated agarose solutions

 

Change Country | Contact Us | Privacy Policy | Terms and Conditions   ©2009 Calbiochem, Novabiochem, & Novagen are brands of EMD Chemicals Inc., an Affiliate of Merck KGaA, Darmstadt, Germany.