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In Cell Biology, no protein can stay isolated within a cell. Protein–protein interactions are very important and necessary to execute cellular functions. The most popular method to find "protein friends" is immunoprecipitation (IP)/ Co-immunoprecipitation (Co-IP). The magnetic beads composed of the superparamagnetic polymer microspheres coupled with ligands, provide a fast, convenient, and efficient method to study protein interaction by IP, Co-IP and pull down.
Where are magnetic beads?
Biological magnetic beads are generally superparamagnetic microspheres with fine particle size (μm) characterized by the properties as following:
1) The superparamagnetism allows magnetic beads to rapidly gather in the magnetic field and disperse uniformly outside the magnetic field. The proper and uniform particle size ensures its strong magnetic reactivity without precipitation.
2) The abundant surface-active groups enable the magnetic beads to bind to biomolecules and separate from the sample to be tested under the action of external magnetic field.
Figure 1. The composition of MCE magnetic bead
A, G, L, A/G represent Protein A, Protein G, Protein Land Protein A/G, respectively; Anti-HA, Anti-Flag, Anti-c-Myc, Anti-GST represent Anti-HA antibody, Anti-Flag antibody, Anti-c-Myc antibody and Anti-GST antibody, respectively.
How to choose the appropriate beads?
Firstly, the magnetic force (magnetization time) and particle size (diameter and homogeneity) should be considered for this study. Secondly, the appropriate surface-active group, the derived coupling method, and the amount of protein binding should be selected according to the experimental purpose.
Taking Protein A/G magnetic beads as an example, the experimental steps of IP are described in detail, and the possible problems and solutions are as follows.
Step1 — The preparation of Antigen samples
The preparation of antigen sample (cell lysate) is the first and critical step of IP experiments. A suitable cell lysate can stabilize the natural structure of proteins, inhibit enzyme activity, minimize the denaturation of antibody binding sites and release protein from cells or tissues. Generally, NP-40, RIPA, Western and/or IP cell lysis buffers can be selected. Cell lysates (also known as antigen samples) should be kept on ice all the time, and protease inhibitors should be added to the lysate to prevent proteolysis.
Of note:
1. Non-ionic lysates such as NP-40 are commonly used to avoid destroying the protein-protein interaction in Co-IP experiments. 2. Ensure that the target protein has a high level of expression in antigen samples.
Figure 2. Culture, collect, lyse cells and release target protein.
Step2 — The binding of samples
You will get the complex consisting of magnetic bead- antibody-antigen sample through this step. The order in which the materials are added has certain influence on the yield of target protein.
Figure 3. The common method for sample binding in IP experiment
All in one—Direct mixing of magnetic beads, antibody and antigen samples together; The direct binding —Binding of antibody to the magnetic beads first and then to the antigen sample; The indirect binding —Binding the antibody to antigen samples first and then to magnetic beads.
The Optional step 1 — The pretreatment of antigen samples: to reduce nonspecific binding, antigen samples can be bound to Protein A/G magnetic beads or isotype control antibodies alone. Tips: Pretreatment is helpful to remove molecules that may be non-specifically bound to antibodies or magnetic beads during the IP process, thus improving the signal-to-noise ratio.
The Optional step 2 — The cross-linking immobilization of antibody: To keep the antibody from co-eluting with the target protein, it can be covalently attached to protein A/G using a cross-linking agent at the time of direct binding. Subsequently the target protein can be eluted by acid elution method. Tips: Direct mixing of magnetic beads, antibody and antigen samples is the fastest, but the purity and yield of the target protein will be very low. With the indirect binding, the yield of target protein is the highest, but there is a chance that the antibody will be eluted together with the protein in the elution step. With the direct binding method, you will also get a higher yield of the target protein which can be eluted separately by cross-linking the antibody. Generally, if the abundance of protein is high, you can choose both because the difference between direct and indirect binding is minimal. However, you should better choose the indirect binding in order to obtain the higher yield when the abundance of target protein is not as high as expected.
The choice of binding/washing buffer in the binding step is also of great importance. Generally, the binding/washing buffer can be the same, such as TBST/PBST. In addition, detergent (e.g.0.5-1% NP-40/TritonX-100/Tween-20), salt ion concentration (e.g. 250 mM NaCl/1-2 mM DTT/β-ME) and increased washing time can be added to improve the % purity of the eluted protein.
Step3 — The elution of antigen
The elution method is selected according to the following application:
Figure 4. The common method for antigen elution
1) The elution method of denaturation: If Western Blot is used for downstream analysis, elution is usually performed directly using SDS-PAGE loading dye (heating at 95℃ for 5 min), which denatures and reduces the protein for electrophoresis and dissociates the affinity with high efficiency. With this method, however, it also results in the elution of non-targeted molecules such as antibodies, making the obtained proteins biologically inactive.
2) The acid elution method: The glycine with the final concentration of 0.1 M-0.15 M (pH 2.5-3) is the most efficient non-denaturing elution buffer in IP. The low pH uncouples most antibody-antigen interactions, so the eluted sample retains its original biological activity and can be used for the subsequent functional analysis. Nevertheless, acid elution is not a panacea as it cannot dissociate some antibody-antigen interactions or can damage some antibodies that are sensitive to low pH environments.
Of note:
1. Adding a neutralizing solution to the receiver tube prior to elution will alleviate the sensitivity of antibody to low pH environment. To avoid co-elution of the antibody with the target protein, use a cross-linking agent to covalently link the antibody to protein A/G, followed by acid elution.
3) The competitive elution method: If the target protein is tagged, use a higher concentration of tag or ligand for competitive elution. For example, the Flag tag protein can be competitively eluted by 3X Flag peptide. This maintains the original biological activity of the eluted sample without contamination by antibody fragments.
Step4 — The WB verification
1) The signal of the target protein is weak or absent. Is the protein expressed? Is the added sample sufficient? Is there enough incubation time? Is the lysate selected properly? Is the elution method appropriate?
Figure 5. There is weak signal or no signal in IP group. There is a band in input group but there is no band in IP group or the band is weak.
2) High background value or multiple bands a. Optimize protein loading. In IP experiments, overloading of total protein may lead to non-specific binding. It is recommended that the loading amount be about 1 mg. b. Optimize the washing buffer by adding detergent (e.g. 0.5-1% NP-40/Triton X-100/Tween-20), increasing the concentration of salt ions (e.g., 250 mM NaCl/1-2 mM DTT/β-ME), extending the washing time, washing frequency, etc. c. The unnecessary high-intensity elution buffer (e.g., SDS-PAGE loading dye) can cause a variety of non-specific proteins (e.g., Protein A/G or streptavidin subunit) to be co-eluted with antigens. This can be avoided with mild buffers (e.g. 0.1 M glycine, pH 2.5). Antibody contamination: refer to the previous discussion on antibody cross-linking and immobilization or use a different type of primary antibody for IP detection and subsequent WB validation; d. Choose proper protease inhibitor to prevent protein degradation; e. The pretreatment of antigen samples (binding the antigen sample to protein A/G magnetic beads) can reduce some nonspecific binding.
3) The settings for positive and negative control The successful bands are always similar, and the failed are different. When analyzing the results, the negative and positive control would be helpful. The positive control: Confirmation of the target protein (IP) or an interacting protein pair (Co-IP, such as bait protein X and target protein Y) is indeed present in cell lysate, which is often called input. The cell lysate or extracted protein solution can be directly used for Western Blot. The negative control: It is gratifying to detect both target protein and interacting protein pairs after IP/ co-IP, but the negative control is required to confirm that the results are not false positives. Output: In some Co-IP experiments, output group is used for the detection of bait protein X and target protein Y obtained from supernatant during WB verification.
Hope this article can help you to get the desired results during protein interaction studies by IP, Co-IP and pull down.
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