Immunoprecipitation involves the interaction between a protein and its specific antibody, the separation of these immune complexes with Protein A, and the subsequent analysis by SDS-PAGE. This technique provides a rapid and simple means to separate a specific protein from whole cell lysates or culture supernatants. Additionally, one can use immunoprecipitation to confirm the identity or study biochemical characteristics, posttranslational modifications, and expression levels of a protein of interest. The procedure can divided into several stages:
1 Sample preparation
2 Preclearing
3 Antibody incubation/formation of antibody-antigen complexes
4 Precipitation
5 Analysis by SDS-PAGE
The protocol below offers a general guideline for immunoprecipitation, optimization may be required for each specific antigen and antibody. The abundance of a given protein n a sample is variable and a critical factor for obtaining desired results. In this protocol, a cell concentration of approximately 10(7) cells/ml is recommended as starting point. Fine adjustments to cell concentrations and volumes of cell lysate used in each immunoprecipitation experiment may be necessary.
The choice of lysis buffer is critical and dependent on the nature of the protein to be studied. NP-40, a non-ionic detergent, is the most commonly used detergent in cell lysis buffers. Increasing the salt concentration, decreasing the detergent concentration, or changing the detergent to Triton X-100, Saponin, Digitonin, CHAPS or others are steps that can be taken to optimize conditions for immunoprecipitation.
The preclearing step is incorporated into the procedure to lower the amount of non-specific contaminants in the cell lysate and to remove proteins with high affinity for Protein G or Protein A. Many investigators choose to skip this cases where the protein of interest exists abundantly in the sample.
The success of immunoprecipitation depends on the affinity of the antibody for its antigen as well as for Protein G or Protein A. In general, while polyclonal antibodies are best, purified monoclonal antibodies (mAb), ascites fluid, or hybridoma supernatant can also be used. Some mAbs work very well, whereas others may give unsatisfactory results. It is recommended that the specified experimental conditions be followed as published, however, optimization of condition by the investigator may be necessary. The strength of interaction between the mAb and Protein A is an important factor in the decision of which slurry to use. Protein G coupled to some insoluble matrix (e.g. sepharose beads) binds well to most subclasses of rat immunoglobulins and mouse lgG1, while Protein A binds much better to mouse IgG2a, IgG2b, and IgG3.
Step I: Cell Lysate Preparation
Harvest approximately 10(7) cells.
The total number of cells per ml and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody.
Wash cells with~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes.
Discard supernatant and repeat step2.
If using tissue culture dish adherent monolayer cells, wash cells in the flask two times with room temperature PBS and aspirate without disturbing the monolayer.
After the second wash, remove supernatant completely and resuspend the cell pellet in 1ml of cold Lysis. Buffer containing 1X Protease Inhibitor Cocktail (final concentration of 10(7) cells/ml). Gently vortex the tube.
To prevent protease action effectively, the Lysis Buffer should be pre-chilled and all the following steps should be kept in the cold. The Protease Inhibitor Cocktail should be added to the required volume of the cold Lysis Buffer just before use. For adherent monolayers, add 1ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail per 100mm culture dish.
Place the tube or the dish on ice for 30 minutes, with occasional mixing.
Spin cell lysate at 10,000xg for 15 minutes at 4 degree Celsius.
Carefully collect supernatant, without disturbing the pellet and transfer to a clean tube. The cell lysate can be frozen at this point for long-term storage at -80 degrees C. Discard pellet.
Step II: Cell Lysate Preclearing
Transfer 50μl of the Protein G beads slurry (commercially available from several vendors) to an eppendorf tube and add 450μl cold Lysis Buffer. Spin at 10000xg for 30 seconds and remove the Lysis Buffer. Wash one more time with 500μl of cold Lysis Buffer. Resuspend the beads in 500μl of cold Lysis Buffer.
Add this 50μl of prepared Protein G slurry and 500μl of Cell Lysate to eppendorf tube and incubate on ice for 30-60 minutes.
Spin at 10000xg for 10 minutes at 4 degrees C and transfer the supernatant to a fresh eppendorf. If any bead has been transferred, spin again and carefully transfer the supernatant to another fresh eppendorf tube.
Step III: Immunoprecipitaton
Add 5~10μg of antibody to the eppendort tube containing the cold precleared lysate.
This concentration of mAb is suggested as a starting point. Each investigator may desire to titrate the concentration of antibody and volume of cell lysate in preliminary experiments to extensively determine the optimal conditions.
Incubate at 4 degrees C for 1 hour.
Add 50μl of washed Protein G slurry in prechilled Lysis Buffer (prepared as instructed in Preclearing Step 1 above).
Incubate for 1 hour at 4 degrees C on a rocking platform or a rotator.
Spin the eppendorf tube at 10000xg for 30 seconds at 4 degrees C.
Carefully removes supernatant completely and wash the beads 3~5 times with 500μg of Lysis Buffer.
To minimize background, care should be given to remove the supernatant completely in these washes.
After the last wash, aspirate supernatant and add 50 ug of 1x Laemmli sample buffer to bead pellet.
Vortex and heat to 90~100 degrees C for 10 minutes.
Spin at 10000xg for 5 minutes, collect supernatant and load onto the gel.
Supernatant samples can be collected and kept frozen at this point, if the get is to be run later.
Follow manufacturer’s instructions for SDS-PAGE. Stain the gel for visual analysis of the immunoprecipitated protein.
Antibody used for the immunoprecipitation portion must not be the same antibody used for the western blot portion. A different clone with the same specificity needs to be used.
Immunoprecipitation Buffers:
*NP-40 Cell Lysis Buffer
50 mM Tris-HCI pH 8.0
150 mM NaCl
1% NP-40
*Protease Inhibitor Cocktail (100X)
PMSF, 5mg (50μg/ml)
Aprotinin, 100ug (1μg/ml)
Leupeptin, 100ug (1μg/ml)
Pepstatin, 100ug (1μg/ml)
100% Ethanol bring up to 1ml, aliquot and keep at minus 20 degrees C.
