Proteins often work together in a complex system, rather than acting alone. Accumulating evidence indicates that protein-protein interactions play crucial roles in various biological processes within living cells. co-IP is a widely accepted technique for confirming or detecting protein-protein interactions in vivo. Co-IP experiments allow the identification of proteins through direct or indirect interactions or as part of protein complexes. In this article, a protocol for co-immunoprecipitation (Co-IP), in which an antibody is used to isolate its target antigen and its binding partner from a mixed sample, is presented. Binding partners purified with immunoprecipitated proteins can be identified by western blotting or mass spectrometry.
Creative Proteomics provides custom experimental design and refine Co-IP parameters for your protein and protein interactions (PPIs) research.
How to Conduct A Co-IP
1. Sample Preparation
The first step in the Co-IP protocol is to prepare the sample to be analyzed, which can be derived from cells, tissues, or other biological sources. Proper sample preparation is critical to ensure efficient immunoprecipitation and accurate detection of protein complexes.
Sample preparation involves lysing cells or tissues to release the protein of interest. Lysis buffers containing detergents, salts, and protease inhibitors are commonly used to disrupt cell membranes, solubilize proteins and prevent their degradation. The choice of lysis buffer should be optimized based on the nature of the sample and the desired downstream application.
2. Antibody Incubation
Subsequently, the sample is subjected to an incubation process in the presence of particular antibodies, which can either be monoclonal or polyclonal, contingent on the experimental prerequisites. Prior to usage, the antibodies ought to undergo validation and optimization to ascertain their specificity and affinity towards the target protein. The sample is gently stirred during this incubation stage, enabling the formation of antibody-protein complexes. To achieve efficient binding between the antibodies and their respective target proteins, it is essential to optimize the temperature and duration of incubation.
3. Immunoprecipitation
After the antibody incubation, the immunoprecipitation step is performed to selectively isolate the protein complexes of interest. This is achieved by adding a suitable matrix to capture the antibody-protein complexes. Commonly used matrices include Protein A or Protein G agarose beads, which bind to the Fc region of the antibodies.
The sample, along with the antibody-protein complexes, is incubated with the matrix under gentle agitation to allow for efficient binding. The matrix is then collected by centrifugation or filtration, and the unbound proteins and contaminants are washed away.
4. Elution
Once the protein complexes are captured on the matrix, the next step is to elute the bound proteins for further analysis. Elution is typically performed by disrupting the antibody-protein interactions using specific elution buffers. These buffers can contain low pH, chaotropic agents, or competitive binding agents, depending on the desired conditions.
The eluted protein complexes are collected, and the elution process can be repeated if necessary to maximize the recovery of the target proteins and their interacting partners. The elution conditions should be optimized to ensure the preservation of the protein complexes and their structural integrity.
5. Analysis
The Co-IP protocol concludes with analyzing the eluted protein complexes using techniques like Western blotting, mass spectrometry, or functional assays. Western blotting confirms protein-protein interactions by detecting and quantifying specific proteins with antibodies. Mass spectrometry can identify the proteins in the complexes, giving a comprehensive view of the interaction network and potential new partners. Functional assays assess the biological relevance of the complexes and provide insight into their roles. Careful optimization, control experiments, independent validation, and experiment replication are necessary for reliable and reproducible results. Negative controls like non-specific antibodies or IgG isotype controls help exclude non-specific interactions.
Detailed Steps for Co-IP Experiment
Preparation:
1. Pre-chilled PBS, RIPA Buffer, cell scraper (wrapped in plastic wrap and buried in ice), centrifuge.
2. Wash cells twice with pre-chilled PBS, and remove the final PBS wash completely.
3. Add pre-chilled RIPA Buffer (1 ml/10^7 cells, 10 cm dish or 150 cm^2 flask; 0.5 ml/5×10^6 cells, 6 cm dish or 75 cm^2 flask).
4. Use a pre-chilled cell scraper to scrape cells off the dish or flask, transfer the suspension to a 1.5 ml Eppendorf tube, and shake gently at 4°C for 15 minutes (place the tube on ice and shake horizontally on a shaker).
5. Centrifuge at 4°C, 14,000 g for 15 minutes, and immediately transfer the supernatant to a new centrifuge tube.
6. Prepare Protein A agarose by washing the beads twice with PBS, then prepare a 50% concentration in PBS. It is recommended to remove the tip of the pipette to avoid damaging the agarose beads during handling.
7. Add 100 μl of Protein A agarose (50%) to every 1 ml of total protein, and shake gently at 4°C for 10 minutes (place the tube on ice and shake horizontally on a shaker) to remove non-specific proteins and reduce background.
8. Centrifuge at 4°C, 14,000 g for 15 minutes, and transfer the supernatant to a new centrifuge tube, removing the Protein A beads.
9. Prepare a protein standard curve using the Bradford method to determine protein concentration. Dilute the total protein at least 10-fold before measurement to reduce the interference of detergents in the cell lysis buffer (quantify and aliquot, can be stored at -20°C for one month).
10. Dilute the total protein to approximately 1 μg/μl with PBS to reduce the concentration of detergents in the lysis buffer. If the target protein is present at a low level in the cells, the total protein concentration should be slightly higher (e.g., 10 μg/μl).
11. Add a certain volume of rabbit antibody to 500 μl of total protein. The dilution ratio of the antibody depends on the abundance of the target protein in different cell lines.
12. Shake the antigen-antibody mixture slowly at 4°C overnight or at room temperature for 2 hours. For kinase or phosphatase activity analysis, incubation at room temperature for 2 hours is recommended.
13. Add 100 μl of Protein A agarose to capture the antigen-antibody complexes, and shake the mixture slowly at 4°C overnight or at room temperature for 1 hour. If the antibody used is mouse or chicken, it is recommended to add 2 μl of "secondary antibody" (rabbit anti-mouse IgG or rabbit anti-chicken IgG).
14. Centrifuge at 14,000 rpm for 5 seconds, collect the agarose bead-antigen-antibody complexes, remove the supernatant, wash three times with pre-chilled RIPA buffer (800 μl per wash). Sometimes RIPA buffer can disrupt the internal binding of agarose bead-antigen-antibody complexes, so PBS can be used instead.
15. Resuspend the agarose bead-antigen-antibody complexes with 60 μl of 2× loading buffer and mix gently. The volume of the buffer depends on the amount of sample to be loaded (60 μl is sufficient for three lanes).
16. Boil the samples for 5 minutes to denature the free antigen, antibody, and beads. After boiling, centrifuge the samples briefly to collect the remaining agarose beads. The supernatant can be used for electrophoresis, or it can be temporarily frozen at -20°C for future use. Before electrophoresis, the supernatant should be boiled again for 5 minutes to ensure denaturation.
Preparation of RIPA Buffer
Basic components: Tris-HCl (buffer component to prevent protein denaturation), NaCl (salt component to prevent non-specific protein aggregation), NP-40 (non-ionic detergent for protein extraction, prepared as a 10% stock solution in water), sodium deoxycholate (ionic detergent for protein extraction, prepared as a 10% stock solution in water and stored in a light-protected container).
Note: When preparing kinase (enzyme) experiments, do not add sodium deoxycholate, as the ionic detergent can denature the enzyme and result in loss of activity.
RIPA Protease Inhibitors: Phenylmethylsulfonyl fluoride (PMSF, prepared as a 200 mM stock solution in isopropanol and stored at room temperature), EDTA (calcium chelator, prepared as a 100 mM stock solution in water, pH 7.4), leupeptin (prepared as a 1 mg/ml stock solution in water and stored at -20°C), aprotinin (prepared as a 1 mg/ml stock solution in water and stored at -20°C), pepstatin (prepared as a 1 mg/ml stock solution in methanol and stored at -20°C).
RIPA Phosphatase Inhibitors：Activated Na3VO4 (prepared as a 200 mM stock solution in water, see Sodium Orthovanadate Activation Protocol), NaF (prepared as a 200 mM stock solution in water and stored at room temperature).
Note: When preparing phosphatase experiments, do not add phosphatase inhibitors.
Working Solution Preparation:
Prepare 100 ml of modified RIPA buffer.
Weigh 790 mg of Tris base and add it to 75 ml of deionized water. Add 900 mg of NaCl and stir until completely dissolved. Adjust the pH to 7.4 with HCl.
Add 10 ml of 10% NP-40.
Add 2.5 ml of 10% sodium deoxycholate and stir until the solution is clear.
Add 1 ml of 100 mM EDTA. Bring the volume up to 100 ml with a volumetric flask and store at 2-8°C.
Ideally, protease and phosphatase inhibitors should be added on the day of use (100 μl each of aprotinin, leupeptin, and pepstatin; 500 μl each of PMSF, Na3VO4, and NaF). However, PMSF is unstable in aqueous solutions and degrades by 50% within 30 minutes, so it should be added immediately before use. Other inhibitor components can remain stable in the solution for 5 days.
Final Concentrations of Components in the Working Solution:
Tris-HCl: 50 mM, pH 7.4
NP-40: 1%
Sodium deoxycholate: 0.25%
NaCl: 150 mM
EDTA: 1 mM
PMSF: 1 mM
Aprotinin, leupeptin, pepstatin: 1 μg/ml each
Na3VO4: 1 mM
NaF: 1 mM
Experiment Process
1. After 24-48 hours of transfection, harvest the cells and add an appropriate amount of cell lysis buffer (containing protease inhibitors). Keep the lysate on ice and incubate for 30 minutes. Centrifuge the cell lysate at maximum speed for 30 minutes at 4°C and collect the supernatant.
2. Take a small aliquot of the lysate for Western blot analysis. Add 1 μg of the corresponding antibody to the remaining cell lysate and incubate overnight at 4°C with gentle shaking.
3. Take 10 μl of protein A agarose beads and wash them three times with an appropriate amount of lysis buffer, centrifuging at 3,000 rpm for 3 minutes each time.
4. Add the pre-treated 10 μl of protein A agarose beads to the cell lysate incubated with the antibody and incubate at 4°C with gentle shaking for 2-4 hours to allow the antibody to bind to the protein A agarose beads.
5. After the immunoprecipitation reaction, centrifuge the mixture at 3,000 rpm for 3 minutes at 4°C to pellet the agarose beads. Carefully remove the supernatant and wash the agarose beads with 1 ml of lysis buffer for 3-4 times. Finally, add 15 μl of 2× SDS loading buffer and boil for 5 minutes.
6. Perform SDS-PAGE, Western blotting, or mass spectrometry analysis to determine the interacting protein through immunoprecipitation.
7. Wash suitable cells on 30 plates (10 cm) with phosphate buffer. Scrape the cells from each plate into 1 ml of cold EBC lysis buffer.
8. Transfer each milliliter of cell suspension to microcentrifuge tubes and centrifuge at maximum speed for 15 minutes at 4°C.
9. Collect the supernatant (approximately 30 ml) and add 30 μg of the appropriate antibody. Shake the immunoprecipitation mixture at 4°C for 1 hour.
10. Add 0.9 ml of protein A-Sepharose suspension and shake the immunoprecipitation mixture at 4°C for 30 minutes.
11. Wash the protein A-Sepharose mixture with NETN buffer containing 900 mM NaCl, repeat the washing step 5 times, and finally wash once with NETN buffer.
12. Remove the liquid portion of the mixture and add 800 μl of 1× SDS sample buffer to the beads. Boil for 4 minutes.
13. Load the sample onto a large-pore gradient SDS-PAGE gel and electrophoresis overnight at a constant current of 10 mA.
14. Visualize protein bands by Coomassie blue staining.
15. Cut out the target band from the gel and transfer it to a microcentrifuge tube. Wash it twice with 1 ml of 50% acetonitrile for 3 minutes each time.
16. Digest the proteins in the gel with trypsin and then perform peptide elution.
17. Separate the peptides by narrow-bore high-performance liquid chromatography. Perform automated Edman degradation sequencing of the collected peptides on an ABI 477A or 494A machine.
Points to note
(1) Cell lysis should be performed under gentle conditions to avoid disrupting protein-protein interactions within the cells. Non-ionic detergents such as NP40 or Triton X-100 are commonly used. The lysis conditions may vary for different cell types and should be determined based on experience. High concentrations of denaturing agents like 0.2% SDS should be avoided, and various enzyme inhibitors should be added to the cell lysis buffer.
(2) Specific antibodies should be used, and multiple antibodies can be used together.
(3) Control antibodies should be used. For monoclonal antibodies, normal mouse IgG or another unrelated monoclonal antibody can be used as a control. For polyclonal antibodies raised in rabbits, normal rabbit IgG can be used as a control.
To ensure the reliability of results in immunoprecipitation experiments, the following points should be noted:
(1) Ensure that the precipitated proteins are specifically obtained through the added antibodies and not from non-specific sources. The use of monoclonal antibodies can help avoid contamination.
(2) Confirm the specificity of the antibodies, ensuring that they do not precipitate proteins in cell lysates that do not express the antigen.
(3) Determine that the protein-protein interactions occur within the cells and are not solely due to cell lysis. This may require protein localization studies to confirm.
Co-IP Protocol Optimization
A successful Co-IP experiment requires careful optimization of various parameters. Factors such as antibody concentration, incubation time, and washing conditions need to be optimized to ensure specific and efficient capture of the protein complex of interest. Experimental conditions may vary depending on the nature of the target protein, the sample source, and the intended downstream analysis techniques.
Creative Proteomics offers comprehensive Co-IP protocols and technical support to guide researchers in optimizing their experiments and obtaining reliable results. These protocols provide step-by-step instructions for sample preparation, antibody selection, immunoprecipitation, washing steps, elution, and subsequent analysis.
Antibody Selection
One of the critical factors in Co-IP protocol optimization is the selection of appropriate antibodies. The choice of antibodies should be based on their specificity, affinity, and compatibility with the experimental system. It is essential to use antibodies that specifically recognize the target protein and efficiently immunoprecipitate it along with its interacting partners. Commercially available antibodies or those generated in-house can be used, and it is recommended to validate their specificity using techniques like Western blotting or immunofluorescence.
Pre-clearing Step
To enhance the specificity of the Co-IP experiment and minimize false-positive outcomes, the protocol typically includes a pre-clearing step aimed at reducing non-specific binding and background signals. Such a step involves treating the protein lysate with an appropriate control antibody or beads, which effectively removes non-specifically binding proteins.
Lysis Buffer Composition
The composition of the lysis buffer plays a crucial role in maintaining protein-protein interactions and preventing protein degradation during the Co-IP procedure. The lysis buffer should contain appropriate detergents to solubilize the proteins, as well as protease and phosphatase inhibitors to prevent protein degradation and preserve protein phosphorylation states. The choice of lysis buffer should be optimized for the specific protein complex and cell/tissue type under investigation.
Incubation Conditions
Efficient binding between antibody and antigen and the formation of stable immunocomplexes greatly depends on the incubation conditions during the Co-IP process. Optimal antibody-antigen interaction can only be achieved through the optimization of the incubation time and temperature. It is advisable to conduct preliminary experiments to determine the ideal incubation conditions tailored to the specific antibodies and protein complex under investigation.
Washing Steps
Thorough washing steps are crucial to remove non-specifically bound proteins and contaminants, thereby increasing the specificity of the Co-IP experiment. The washing conditions, including the buffer composition, pH, and number of washes, should be optimized to achieve efficient removal of unwanted proteins while retaining the desired protein complex.
Elution of Immunocomplexes
The elution step is performed to release the immunocomplexes from the antibody-protein A/G bead complexes for downstream analysis. Different elution methods can be employed, such as low-pH elution, competitive elution using excess antigen peptide, or direct elution using denaturing agents. The choice of elution method should be optimized to achieve efficient recovery of the immunocomplexes while maintaining their integrity.
Validation and Controls
Including suitable positive and negative controls is critical for guaranteeing the trustworthiness of Co-IP outcomes. Known protein-protein interactions that have been verified before make excellent positive controls. Negative controls, such as non-specific antibodies or isotype-matched IgG, are helpful in determining the specificity of the immunoprecipitation method. Moreover, techniques like Western blotting or mass spectrometry can be utilized to confirm the Co-IP results.
Troubleshooting and Optimization
Despite careful optimization, Co-IP experiments may encounter challenges and produce suboptimal results. In such cases, troubleshooting steps can be taken to identify and address the issues. Troubleshooting may involve adjusting antibody concentrations, optimizing washing conditions, modifying the lysis buffer composition, or exploring alternative elution methods. Iterative optimization is often necessary to fine-tune the Co-IP protocol for specific experimental requirements.
Quality Control and Reproducibility
In order to assure the dependability and consistency of Co-IP experiments, quality control measures must be incorporated in the entire protocol. This involves evaluating the integrity of protein samples, verifying the effectiveness of immunoprecipitation, and authenticating the existence of the target protein and its interacting partners.
An established technique for validating Co-IP outcomes is carrying out Western blot analysis. Using specific antibodies to probe the immunoprecipitated proteins, scientists can confirm the existence of the target protein and its interacting partners. This step is vital for confirming the specificity of the Co-IP experiment and ascertaining that the observed interactions are not as a result of non-specific binding.
Another quality control measure is the inclusion of negative controls where appropriate. Negative controls can help in identifying and exempting non-specific interactions or background signals. Isotype-matched control antibodies or non-specific IgG can serve as negative controls in evaluating the specificity of immunoprecipitation.
Reproducibility is a fundamental component of every scientific experiment, including Co-IP. To ensure reproducibility, it is advisable to carry out independent replicates of the Co-IP experiment. This helps in evaluating the consistency of the results and establishing whether the observed protein interactions are sturdy and trustworthy.
Optimization for Specific Applications
The optimization of the Co-IP protocol may vary depending on the specific application or research question. For example, if the aim is to study transient protein interactions, the timing of the Co-IP experiment may need to be optimized to capture these interactions at the appropriate time points. Additionally, the choice of cell or tissue type, as well as the expression level of the target protein, may influence the optimization process.
Moreover, the downstream applications following Co-IP, such as mass spectrometry or functional assays, may require additional optimization steps. For mass spectrometry-based analysis, special considerations need to be taken to ensure efficient protein identification and quantification. This may involve using compatible buffers and reagents that are compatible with mass spectrometry analysis.
Collaboration and Shared Protocols
In the scientific community, collaboration and sharing of protocols are vital for advancing research and ensuring the reproducibility of results. Researchers can benefit from sharing their optimized Co-IP protocols with the scientific community. This promotes transparency, allows for comparisons between different laboratories, and facilitates the standardization of Co-IP protocols across research groups.
Additionally, companies like Creative Proteomics play a significant role in supporting researchers by providing optimized Co-IP protocols and offering technical assistance. Our expertise and experience in protein research can aid researchers in overcoming challenges and achieving reliable results in Co-IP experiments.
In conclusion, the optimization of the Co-IP protocol is crucial for obtaining reliable and reproducible results. By carefully considering factors such as antibody selection, lysis buffer composition, incubation conditions, washing steps, elution methods, and validation controls, researchers can enhance the specificity and sensitivity of the Co-IP experiment. Moreover, optimization can be tailored to specific applications and research questions, ensuring the protocol's suitability for the intended downstream analysis. Collaboration, shared protocols, and support from companies like Creative Proteomics further contribute to the advancement of Co-IP techniques and their successful application in protein research.
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