Troubleshooting CO-IP Assays: Common Problems and Effective Solutions

Co-IP assays (Protein immunoprecipitation, Immunoprecipitation methods) are similar to immunoprecipitation assays in that they are both proteins that detect protein-protein interactions. Co-IP technology is used to analyze whether there is an interaction between two or more proteins, based on immunoprecipitation experiments, Compared with other protein-protein interaction experimental techniques (GST Pull-down, Western Blot, etc.), Co-IP technology has higher specificity, sensitivity, and high repeatability, which can avoid the interference of non-specific binding and reflect the interaction of proteins in the natural state.

Protein immunoprecipitation have the following advantages: 1. It can provide evidence directly for protein-protein interactions that enable precise targeting of interacting proteins, and direct capture of specific protein complexes from cell lysates. 2. To ensure the natural state of the protein complex, and to provide more real biological information for research. 3. It can provide great flexibility and a wide range of research applications that Can be applied to specific tissue cells or whole organisms.

Immunoprecipitation methods are currently used in disease mechanism research, drug development, biomarker discovery, etc. It provides a new target for disease diagnosis and treatment, and the interaction of disease-related proteins can be revealed; Understanding the molecular mechanism of drug action accelerates the process of new drug development; It provides the possibility for early diagnosis of diseases that plays a key role in the discovery of biomarkers.

Because the Co-IP assays will have different degrees of problems from sample preparation to development, which need to pay attention to a lot of experimental details, common protein immunoprecipitation experiment problems mainly include the following aspects:

Problem 1: The Destination Strip Is Not Detected Or The Signal Is Weak

Reason analysis:

1. Low or no expression of the target protein

Solution: The protein expression can be verified before the experiment, or the sample size can be increased; The protein lysate added to IP was increased and pre-treated.

2. The recognition epitopes of target proteins are blocked by interacting proteins

Solution: Select antibodies with high affinity.

3. The target protein is not eluted

Solution: Select the right strength and pH eluent,

4. the target protein is lost in the washing process

Solution: Add appropriate detergent and NaCl.

5. Degradation of target protein

Solution: Add protease inhibitors when handling samples, and all operations are carried out on ice to avoid repeated freezing and thawing of protein samples.

6. Selection of antibodies

Solution: Replace the antibody or change the amount of antibody used to eliminate the problem of antibody failure.

Problem 2: The Background Signal Is Too High

Reason analysis:

1. Degradation of target protein

Solution: Add protease inhibitors when handling samples, and all operations are carried out on ice to avoid repeated freezing and thawing of protein samples.

2. The sample contains protein complexes

Solution: Short ultrasonic sample, centrifuge and take supernatant for follow-up experiment.

3. Washing is not thorough

Solution: Increase detergent and NaCl in the washing solution.

4. Non-specific proteins bind to magnetic beads

Solution: Pre-treat with 1-5% BSA before use.

5. the antibody concentration is too high

Solution: Select the appropriate antibody and adjust it to the appropriate concentration.

6. Poor specificity of antibody

Solution: Replace the antibodies.

Problem 3: False Positives

Reason analysis:

The phenomenon of non-specific binding (between magnetic beads, antibodies, and proteins) appears during the experiment.

Solution: Set up multiple control groups to exclude non-specific interference during verification.

Problem 4: CO-IP Verified That The Two Proteins Interact, While Other Experiments Verified The Opposite Results

Reason analysis:

CO-IP is one of the technical means to verify protein interaction, and the corresponding results weren’t verified by other technical means, which may be caused by the presence of intermediate proteins involved in the interaction of CO-IP.

Problem 5: CO-IP Verified That The Two Proteins Don’t Interact With Each Other, While Other Experiments Verified The Opposite Results

Reason analysis:

Co-IP assays could not detect weakly interacting or low expression protein.

Problem 6: The Input Stripe Is clear, While The Co-IP Stripe Is Weak

Reason analysis:

If the interaction between two proteins is weak, or others are competing with the target protein.

Solution: The decoy protein can be re-tested as a target protein to increase the chance of interaction between the two proteins.

Problem 7: There Are No Stripes For Input, But Stripes For IP

Reason analysis:

The protein may be degraded or the content is low.

Solution: A protease inhibitor is added to the sample while it is being processed, all on ice to prevent protein degradation and in addition， suggested that the loading amount of the input group be increased to ensure the accuracy and reliability of experimental results.

Problem 8: Neither Input Nor IP Has Stripes

Reason analysis:

Protein degradation or low content.

Solution: Add protease inhibitors when handling samples, and increase the sample quantity.

Based on the above, KMD Bioscience can provide various sample processing services and corresponding Co-IP technical services, compared with other biological companies, KMD Bioscience's Co-IP technology comes from the IPS laboratory of Kyoto University in Japan, which can provide high-quality Co-IP technical services to promote the smooth progress of your research or project.

In addition, KMD Bioscience also provides protein-protein interaction services, including yeast two-hybrid, pull-down, immunoprecipitation assays, etc., to meet the experimental needs of different customers.

References:

[1] Pillai-Kastoori L, Heaton S, Shiflett SD, et al. Antibody validation for Western blot: By the user, for the user [J]. J Biol Chem. 2020, 295(4): 926-939.

[2] Ferguson RE, Carroll HP, Harris A, et al. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies [J]. Proteomics. 2005, 5(2): 566-571.

[3] Ren G, Juhl M, Peng Q, et al. Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells [J]. Immunol Cell Biol. 2022, 100(10): 822-829.