How much sample volume do I need to prepare for CoIP?
The CoIP experiment needs to ensure sufficient protein concentration to maintain the originally existing protein interactions, especially when the protein abundance is low, the interaction force is not strong, or when using tissues that are not easy to extract proteins. Therefore, cells >2x107, animal tissues >500mg and plant tissues >2g should be prepared for CoIP experiments, and more attention should be paid to storage at -80°C after collection and avoid repeated freezing and thawing.
What about CoIP protein degradation?
- Pay attention to the operation on ice
- Add protease inhibitor and phosphatase inhibitor
Why are the target bands detected in both IgG and IP groups?
The results of CoIP-WB identification show that there is no target protein band in IgG group, but there is a target protein band in IP group, which means the IP process is successful. It is extremely unlikely that the mass spectrometry will identify the target band in the IgG group. If this is the case, it is possible that the crosstalk occurred during the gel cutting and mass spectrometry sample processing, which has no effect on the conclusion of the reciprocal proteins in the IP group.
Why do two proteins interact with each other by Co-IP, but not by other validation methods? Is Co-IP unreliable?
Co-IP can verify the relationship between two proteins interacting with each other. It is possible that the lack of interaction with other validation methods is due to the presence of intermediate proteins involved in the interaction in Co-IP.
Why other methods can verify the interaction between two proteins, but not with Co-IP?
Co-IP is not able to detect weak interactions or protein expressions that are too low.
Why the protein interacting with the target protein is not detected in the Co-IP experiment or the signal detected is too weak?
It is possible that the interaction between protein and protein is too weak or unstable.
What does input/IP/IB in Co-IP refer to respectively?
Input refers to the total protein, which is a positive control; IP is the precipitation of the corresponding protein with the corresponding antibody; IB refers to the reciprocal signal.
What are the antibody requirements for the Co-IP assay?
For self-protein antibodies, the antibody needs to be of IP grade and needs to be tested for potency. Commercially available labeled antibodies also need to be of IP grade.
How to avoid false positives in the Co-IP assay?
(1) If the antibody concentration is high, reduce the antibody concentration.
(2) If the antibody specificity is poor, replace the antibody.
(3) If the protein abundance is very high, increase the number of washes.
What is the role of ProteinA/G agarose beads in Co-IP?
ProteinA/G binds specifically to FC fragments of immunoglobulins and therefore binds to antibodies. The antibody binds to the target protein and the target protein binds to the interacting protein.
What are the control antibodies used in the Co-IP experiments?
- Monoclonal antibodies: normal mouse IgG or another type of monoclonal antibody.
- Polyclonal antibodies: normal rabbit IgG.
- If it is a labeled antibody use the corresponding labeled protein as a control.
What is the significance of the negative control in the Co-IP experiment?
To exclude the binding of non-specific proteins.
How to remove the effect of heavy and light chains in Co-IP experiments?
It can be effectively avoided with current commercial nanobodies, or excluded with specific secondary antibodies during WB.
What are the reasons that no protein interacting with the target protein is detected in Co-IP or the signal detected is too weak?
(1) The concentration of detergent in the lysate is too high or the formulation is too drastic
Reduce the concentration of detergent or change the type of detergent.
(2) Affected by the subcellular localization of the protein
Re-select the lysate formulation to release the target protein.
(3) Protein-protein interactions are too weak or not too stable
Select antibodies with higher affinity to capture more of the target protein and thus more interacting proteins.
(4) Overexpression to increase the content of the target protein
Select samples with high content of target or interacting proteins for immunoprecipitation experiments.
What should I do if the Co-IP samples are degraded by proteases?
Add a protease inhibitor and keep all operations below 4°C on ice and prevent freeze-thaw.
What should I do if the concentration of Co-IP antibody is too low?
Adjust the concentration of IP and/or IB antibodies and establish a concentration gradient if necessary to find the optimal concentration.
What should I do if the antibody affinity is too low?
Select the appropriate antibodies for IP and/or IB.
What if the IP antibody does not bind to the agarose beads?
Select the appropriate beads for IP and store them properly to prevent deterioration or drying.
What if the Tag is not exposed on the surface of the fusion protein conformation?
Change the tag fusion expression site.
What if the lysate stringency is too high?
Change the lysis solution to a low stringency lysis solution.
What is the difference between IP and Co-IP?
IP: It is a method to purify a protein by directly extracting the target protein with this antibody in the case of known antibodies to a protein.
Co-IP: It is mainly used to detect protein-protein interactions. The final end product is multiple protein complexes.
Why the target protein is not identified by mass spectrometry when the target protein is identified by WB after IP?
Failure to identify the target protein by mass spectrometry is usually due to the low relative abundance of the protein itself in the sample. Other relatively high abundance proteins may mask some of the signal during the mass spectrometry process, resulting in low abundance proteins not being identified in the sample.
What is the criterion for a successful Co-IP experiment?
Input with a target band, negative control without a target band, and reciprocal group with a target band indicate a successful Co-IP.
What other analytical experiments can be performed downstream of Co-IP?
The samples obtained by Co-IP can be used to verify the presence of a specific protein (i.e., the reciprocal protein) by WB with the help of specific antibodies. Protein profiling can also be used to identify the interacting proteins and to search for unknown interacting proteins.
