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Immunoprecipitation Protocols

Immunoprecipitation of Proteins by Conventional Method

Immunoprecipitation is a widely used method to purify specific proteins from complex samples such as cell lysates or extracts. Conventional IP protocols use Protein A or G coupled to an insoluble resin, such as agarose beads, to capture an antigen: antibody complex in solution. The complex is then “precipitated” by centrifugation. Limitations of conventional IP include sample handling and processing difficulties, the inability to release native antigen from the beads for functional assays and poor reproducibility and recovery due to multiple wash steps.

• Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash non-adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells.
• Add ice-cold modified RIPA buffer to cells (1 mL per 107 cells/100 mm dish/150 cm2 flask; 0.5 mL per 5 x 106 cells/60 mm dish/ 75 cm2 flask).
• Scrape adherent cells off the dish or flask with ither a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge tube. Gently rock the suspension on either a rocker or an orbital shaker at 4°C for 15 minutes to lyse cells.
• Centrifuge the lysate at 14,000 xg in a precooled centrifuge for 15 minutes. Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.
• To prepare Protein A or G agarose/sepharose, wash the beads twice with PBS and restore to a 50% slurry with PBS. It is recommended to cut the tip off of the pipette when manipulating agarose beads to avoid disruption of the beads.
• Pre-clear the cell lysate by adding 100 μl of either Protein A or G agarose/sepharose bead slurry (50%) per 1 mL of cell lysate and incubating at 4°C for 10 minutes on a rocker or orbital shaker. Pre-clearing the lysate will reduce non-specific binding of proteins to the agarose or sepharose when it is used later on in the assay.
• Remove the Protein A or G beads by centrifugation at 14,000 x g at 4°C for 10 minutes. Transfer the supernatant to a fresh centrifuge tube.
• Determine the protein concentration of the cell lysate (e.g. if performing a Bradford assay one must dilute the cell lysate at least 1:10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent).
• Dilute the cell lysate to approximately 1 μg/μl total cell protein with PBS to reduce the concentration of the detergents in the buffer. A more concentrated cell lysate (i.e., 10 μg/μl) may be necessary to immunoprecipitate a protein, which is found in low levels in a cell model.
• Add the recommended volume of the immunoprecipitating antibody (see antibody datasheet for detailed information) to 500 μl (i.e., 500 μg) of cell lysate. The optimal amount of antibody that will quantitatively immunoprecipitate the protein of interest should be empirically determined for each cell model.
• Gently rock the cell lysate/antibody mixture for either 2 hours or overnight at 4°C on a rocker or an orbital shaker. A 2 hour incubation time is recommended for the immunopecipitation of active enzymes for kinase or phosphatase assays.
• Capture the immunocomplex by adding 100 μl Protein A or G agarose/sepharose bead slurry (50 μl packed beads) and gently rocking on either a rocker or orbital shaker for either 1 hour or overnight at 4°C. In many instances, immunocomplex capture can be enhanced by adding 2 μg of a bridging antibody (e.g., rabbit- anti-mouse IgG). This is especially important with antibodies, which bind poorly to Protein A, such as mouse IgG1 or antibodies generated in chicken.
• Collect the agarose/sepharose beads by pulse centrifugation (i.e., 5 seconds in the microcentrifuge at 14,000 rpm). Discard the supernatant and wash the beads 3 times with 800 μl ice-cold modified RIPA buffer. Occasionally, washing with modified RIPA buffer will strip the immunocomplex off of the agarose/sepharose beads. In these cases, washing with the milder PBS is recommended.
• Resuspend the agarose/sepharose beads in 60 μl 2x sample buffer and mix gently. This will allow for sufficient volume to run three lanes.
• The agarose/sepharose beads are boiled for 5 minutes to dissociate the immunocomplexes from the beads. The beads are collected by centrifugation and SDS-PAGE is performed with the supernatant. Alternatively, the supernatant can be transferred to a fresh microcentrifuge tube and stored frozen at -20°C for later use. Frozen supernatant should be reboiled for 5 minutes directly prior to loading on a gel.

Conventional Immunoprecipitation Kinase Assay

This protocol is applicable only to kinases whose activity is not altered by cell lysis or immunoprecipitation procedures and that do not require soluble cofactors for activity. Please consult the following resources for additional information:

• Protein Kinase Protocols, edited by Alastair D. Reith, Methods in Molecular Biology, volume 124, Humana Press, 2001 (ISBN 0-89603-700-2).
• Protein Phosphorylation, A Practical Approach, edited D.G. Hardie, Oxford Press, (ISBN 0-19-963729-6).

Verify that the antibody used quantitatively immunoprecipitates the kinase from lysates without affecting its activity, either positively or negatively.

Plan out the necessary components (antibody, buffers, isotope, substrate, and P81phosphocellulose cation-exchange paper/gel lanes) in advance by determining the number of reactions required. Plan on including negative controls (lysate of unstimulated cells; immunoprecipitation with non-immune antibody; sample with no enzyme) as well as a positive control (purified active enzyme). Establish conditions for the kinase assay to ensure that activity is measured well within the linear portion of the reaction (the limiting component should be the availability of active kinase, with substrate and ATP in excess). The most highly active samples are the first to deplete reactants, slowing activity in those samples, and resulting in a compression of measured activities between various samples.

The choice of lysis buffer is extremely important in any immunoprecipitation protocol. The lysis buffer must effectively solubilize the kinase of interest without denaturing it or altering its binding to antibodies. The use of nonionic and/or anionic detergents is used.

Nonionic detergents (e.g., Triton X-100, NP-40) break lipid/lipid and lipid/protein interactions. The anionic detergents (e.g., SDS, Sodium Deoxycholate) are denaturing than nonionic detergents and function to break protein-protein interactions. (Please consult: Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory.Cold Spring Harbor, N.Y.) As a general rule of thumb, to maintain kinase activity, do not use Na Deoxycholate, and for unstable kinases, do not use RIPA buffer EDTA should chelate all cellular Mg2+ and prevent phosphorylation. Phosphorylation can occur following cell lysis, since the cellular pool of ATP serves as the phosphate donor. The Na-vanadate will inhibit most tyrosine protein phosphatases and fluoride should inhibit some serine/threonine specific protein phosphatases.

Protocol to Immunoprecipitate Active Kinase

1. Prepare cell lysate using stimulated cells and perform immunoprecipitation according to procedures and using buffers described in this protocol section.
2. Centrifuge down the immunoprecipitate and remove as much of the supernatant as possible without disturbing the pellet.
   Note:Do not boil the immunoprecipitate after the final washing step. Keep samples on ice.
3. Prepare the solutions for the kinase assay:
   • 5 x assay buffer: 100 mM MOPS; 125 mM ß-glycerol phosphate, pH 7.2; 5 mM EGTA; 5 mM sodium ortho vanadate; 5 mM dithiothreitol (dilute to 1x before use).
   • Magnesium/ATP cocktail: 100 μM non-radioactive ATP and 75 mM magnesium chloride in assay buffer. (If kinase is known to prefer manganese ions for activation, manganese chloride can be substituted formagnesium chloride.)
   • [g32P] ATP: dilute stock solution to final concentration of 1 μCi/μl using magnesium/ ATP cocktail.
   • Substrate (e.g., MBP): prepare a 2 mg/mL solution using assay buffer
4. Prepare reaction buffer mixture by adding 10 μl of each component times the number of assay samples plus one to a single large tube. This will ensure uniformity of the reactants as well as allowing sufficient amounts for all samples. Mix well.
5. Measure the specific activity of the radioactive label by counting 1:10 and 1:100 dilutions of the [g32P] ATP solution in a scintillation counter. Specific activity is expressed as cpm/pmol (the concentration of the radioactive ATP is trivial, so it is assumed to be zero).
6. Dispense the appropriate amount of reaction buffer mixture to each tube containing the immunoprecipitate, still on ice; mix well and transfer all the tubes to a shaking water bath set at the correct temperature. Incubation temperatures and times should be optimized for each kinase.
7. Termination of the assay at the appropriate time can be carried out by adding SDS-sample buffer if the samples are to be electrophoresed, or by returning the tubes to an ice bucket. When dealing with large numbers of samples, it is advisable first to transfer to ice and then to add any termination mix, because of the time between the addition of the termination mix to the first and last tubes.
8. Either spot samples of 30 μl on P81 paper or electrophorese; transfer to nitrocellulose; and expose to film. If using the latter procedure, also excise radioactive bands and determine cpm.
9. Express kinase activity quantitatively using the specific activity of the ATP solution measured above. When assaying a purified kinase, express the catalytic rate in its linear range in mol phosphate transferred from ATP to substrate/min/mg of kinase. Highly active kinases transfer on the order of μmol phosphate/min/ mg of kinase.
10. When assaying kinase activity immunoprecipitated from a lysate, it is not possible to express activity per mg of kinase. Instead, determine the relative activation by subtracting counts incorporated in assays of non-immune immunoprecipitations from gross counts, to determine net cpm. If non-immune cpm are not insignificant, it may be that the immunoprecipitates are contaminated with other kinases. Using net cpm, plot results as fold-activation over unstimulated, or as a percentage of maximal activity.