General Immunoprecipitation Protocol
Licia Miller Product Manager
Immunoprecipitation (IP) is a molecular biology technique based on antigen-antibody specific binding, which is used to selectively separate and enrich target proteins from complex biological samples. The core of this technology is to capture the protein of interest using specific antibodies on a solid support (such as magnetic beads or agarose resin), then remove non-specifically bound impurities by washing, and finally release the purified antigen-antibody complex for downstream analysis.
Immunoprecipitation technology is widely used in protein research, including but not limited to protein-protein interaction research, epitope mapping, post-translational modification analysis, and gene regulation research. For example, immunoprecipitation combined with mass spectrometry can identify and characterize protein complexes and interaction networks in cells; while chromatin immunoprecipitation (ChIP) is used to study the interaction between proteins and DNA, revealing the dynamic changes of chromatin structure and gene expression regulation.
In addition, immunoprecipitation technology can also be integrated with high-throughput screening platforms to systematically analyze protein interaction networks, identify new protein complexes, and reveal signaling pathways behind cellular processes and diseases .
This protocol will introduce the general immunoprecipitation protocol using magnetic beads for cell samples.
Phase 1 Preparation of lysates
The first step is to lyse the cell or tissue sample in a suitable buffer to release the proteins into solution. The ideal lysis buffer will minimize protein denaturation while releasing sufficient protein from the sample.
Nonionic detergents, such as NP-40 and Triton X-100, are less irritating than ionic detergents, such as SDS and sodium deoxycholate. Other variables that may affect the success of immunoprecipitation include salt concentration, divalent cation concentration, and pH.
Required Materials
- Suitable lysis buffer (e.g. N274337)
- Protease inhibitor cocktail (for example: P301903)
- Phosphatase inhibitor cocktail (optional - example: P666104)
- PBS
Experimental Steps
1. Choose an appropriate lysis buffer.
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Keep samples, buffers, and equipment on ice throughout the procedure.
2. Add protease inhibitors to the buffer, including phosphatase inhibitors that phosphorylate proteins.
3. Detach the cells and suspend them in lysis buffer. It is generally recommended to add 300 µL of lysis buffer for 1-3 ×107 cells and 600 µL of buffer for more than 3×107 cells. If the cells cannot be suspended well, increase the lysis buffer appropriately.
4. Incubate the cells with lysis buffer on ice for 10 minutes (without stirring). Then sonicate the lysate 3 times in ice water.
Note: This process may require optimization. If cells are still clumping together, vortex to agitate.
5. Centrifuge at 8,000 × g for 10 min under 4°C, transfer the supernatant to a fresh tube, and place on ice.
6. Determine the protein concentration in the lysate using Bradford or BCA assay.
7. If not used immediately, snap freeze aliquots in liquid nitrogen and store at -80°C.
Phase 2 Immunoprecipitation and washing
There are two main methods for immunoprecipitating proteins. The first method is to mix the antibody with the lysate and then add Protein A/G magnetic beads to the antibody-lysate complex. This method can obtain highly pure proteins; however, the antibody will also elute with the target protein, which sometimes makes it difficult to detect by western blot.
The second method is to prepare antibody- magnetic bead complexes and then incubate them with the lysate to extract the target protein. This method has lower yields than the first method, but avoids the problem of antibody co-elution.
Alternatively, you can perform the IP using the following protocol. If you use an isotype control antibody, you can perform the same experimental procedure and compare it to the IP of your protein of interest when analyzing the results.
Required Materials
- Lysates
- Primary Antibody
- Protein A/G magnetic beads (e.g. P8104 , P8103)
- Magnetic rack
Experimental Steps
1. Incubate the lysate with the antibody overnight at 4°C with gentle agitation.
2. Add Protein A/G magnetic beads to the antibody-lysate complex. Typically, add approximately 100 µL of beads per 1 mL of lysate. Incubate at 4°C with gentle shaking for 1-4 hours.
NOTE: The volumes of lysate and bead solutions may need to be optimized.
3. Wash the magnetic beads three times with PBS buffer to remove unbound impurities .
3.1 Place the tube on the magnetic stand for about 1 minute; the beads should be pulled to the bottom of the tube by the magnet.
3.2 Aspirate and discard the solution, retaining the magnetic beads.
3.3 Add approximately 1 mL of fresh lysis buffer to the magnetic beads and repeat the above steps .
At this point, the target protein should bind specifically to the antibody coated on the beads.
Phase 3 Elution
Elution can be performed under a variety of buffer conditions, including glycine (native), Laemmli (denaturing), and urea buffers.
Of all these buffers, Laemmli buffer containing SDS is the most harsh because it will elute non-covalently bound antibodies and antibody fragments along with the target protein. In contrast, glycine buffer will elute the protein more gently, but the amount of eluted antibody will be reduced.
- Glycine Buffer (Non-denaturing)
Required Materials
- 0.1 - 0.2 M glycine, pH 2.6
- Samples bound to magnetic beads
Experimental Steps
1. Add an equal volume of glycine to the beads and incubate at room temperature for 10 minutes with gentle agitation. The low pH will separate the target protein from the antibody beads.
2. Use a magnetic stand to separate the magnetic beads from the solution. Place the tube on the magnetic stand for about 1 minute so that the magnetic beads are pulled to the bottom of the tube by the magnet.
3. Aspirate the solution, retaining the beads, and transfer the solution to a new tube.
Tips: (1) If necessary, repeat steps 1-3 several times to maximize protein elution.
(2) The beads can be washed with lysis buffer to remove glycine , allowing the beads to be reused.
4. Add Tris-HCl to neutralize the pH of the solution.
NOTE: At this point, aliquot samples, snap freeze, and store at -80°C if not used immediately.
5. Analyze the IP results using Western blotting or other techniques. If an isotype control antibody was used, compare the IP results using the capture antibody to an isotype control antibody.
- Denaturation buffer
Required Materials
- Denaturation buffer: (e.g. 0.1 M DTT, 1× LDS or any other buffer)
- Samples bound to magnetic beads
- Magnetic stand or microcentrifuge
Experimental Steps
1. Add an equal volume of denaturation buffer to the beads and incubate at 100°C for 5 minutes. The high temperature and buffer separate the antibody beads from the target protein and denature the protein at the same time.
2. Use a magnetic stand to separate the magnetic beads from the solution. Place the tube on the magnetic stand for about 1 minute so that the magnetic beads are pulled to the bottom of the tube by the magnet.
3. Aspirate the solution, retaining the beads , and transfer the solution to a new tube.
Note: If not used immediately, aliquot samples, snap freeze, and store at -80°C.
4. Analyze IP results by Western blot. If an isotype control antibody is used, the IP results performed using the capture antibody can be compared with the isotype control antibody.
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