Immunoprecipitation (IP) combined with Western Blot (WB) is currently an important experimental method for studying and validating protein-protein interactions. This article will introduce the experimental procedure of IP-WB and analyze common ideal and abnormal results for everyone.
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The experimental process of IP-WB
The classic IP-WB experiment typically involves the following main steps: firstly, proteins are extracted from cells or tissues using a relatively mild lysis buffer to obtain a protein solution. Then, antibodies and beads are added for incubation, followed by centrifugation or magnetic separation to remove the supernatant containing free proteins, resulting in the formation of a complex containing beads, antibodies, bait proteins, and interacting proteins. After eluting the proteins from the beads, various protein components within the complex are separated by SDS-PAGE based on their molecular weights, transferred to a membrane, and subsequently incubated with primary and secondary antibodies. Finally, the detection signal of the target protein is visualized in the WB results through secondary antibody-mediated imaging.
The specific experimental steps can be divided into:
A. cell lysis;
B. antibody incubation;
C. washing;
D. elution；
E. electrophoresis；
F. membrane transfer;
G. blocking;
H. primary antibody incubation;
I. secondary antibody incubation;
J. imaging, etc.
Among them, the antibodies used for incubation in step B of the IP experiment and the primary antibodies used in step H of the WB experiment are important factors influencing the results of IP-WB. The former targets the bait protein in the IP experiment, while the latter can target either the bait protein or one of its interacting proteins. When evaluating whether the IP experiment has successfully purified or enriched the selected bait protein, the primary antibody used in step H targets the bait protein. When verifying the interaction between the bait protein and its interacting protein, the primary antibody used in step H targets the interacting protein to be validated. The success of the former step is often a prerequisite for the latter experiment.
A) Western blot (WB), immunoprecipitation (IP), and supernatants (SN) of the immunoprecipitation reaction with anti-IL-1IRAcP is shown in the left panel (Oelmann et al., 2015)
Ideal Analysis of IP-WB Results
In an IP-WB experiment, it's standard practice to use the whole-cell lysate as a positive control (input) and a blank IP as a negative control. Therefore, a complete set of IP-WB results should include at least three lanes of WB imaging: Lane 1 for input, Lane 2 for IP, and Lane 3 for IgG/control. For a more thorough evaluation of the IP results, the supernatant after antibody incubation in the IP experiment can serve as an additional negative control for WB detection (Lane 4 for IP-Flowthrough; Lane 5 for IgG/control-Flowthrough).
An ideal IP-WB experiment typically demonstrates the following outcomes:
- Lane 1 (input): This lane usually shows the whole-cell lysate before IP incubation. If bands representing the bait protein are visible here without any other non-specific bands, it indicates normal expression of the bait protein, its recognition by the antibody used, and high specificity in antibody detection. If the input lane includes a mixture of cell lysate, beads, and antibody suspension after IP incubation, bands corresponding to antibody light and heavy chains may also be present.
- Lane 2 (IP): This lane contains the protein sample from the experimental group after antibody incubation, washing, and elution. Strong bands of the bait protein without other non-specific bands suggest successful enrichment and purification of the bait protein by the IP antibody, with high specificity in antibody purification. Typically, non-specific bands from antibody heavy and light chains may appear around 55 kD and 25 kD, respectively.
- Lane 3 (IgG/control): This lane shows the protein sample from the blank control group after antibody incubation, washing, and elution. No bands representing the bait protein should be observed in this lane.
- Lane 4 (IP-Flowthrough): This lane displays the supernatant after antibody incubation in the experimental group. It may show faint bands of the bait protein, indicating incomplete enrichment and purification by the IP antibody.
- Lane 5 (IgG/control-Flowthrough): This lane represents the supernatant after antibody incubation in the control group. If the control group uses blank IgG for IP with the same cells as in Lane 1, it will show bait protein bands similar to Lane 1. If the control group uses cells expressing a blank tag for IP with the same tag antibody as in the experimental group, no bait protein bands will appear in this lane.
Common Issues in IP-WB Results
Appearance of Bait Protein Bands in Control Lane
In the results of an IP-WB experiment, if bands corresponding to the bait protein are visible in both the experimental and control lanes (as shown in the left diagram), there are typically two possible reasons:
a. The molecular weight of the bait protein is close to that of the antibody heavy or light chain, making it indistinguishable, and the bands observed are not solely from the bait protein but also from the antibody heavy or light chains.
b. Improper design or execution of the control group leads to cross-contamination between different samples or adjacent lanes.
The former issue can be addressed by using a different species of primary antibody for WB in step H than the one used for IP incubation in step B, or by using secondary antibodies targeting only the light chain, only the heavy chain, or the complete antibody to eliminate interference from the IP antibody light and heavy chains. The latter requires investigation into the experimental design and steps, followed by repeating the experiment.
Absence or Excessive Bands in the Input Lane
When the input lane shows no bands corresponding to the bait protein, the IP-WB results typically resemble the schematic shown, with no staining of the bait protein in the input lane and only heavy and light chain bands of the antibody in the IP and control lanes. This outcome usually stems from two reasons:
a. Low expression or unsuccessful expression of the bait protein. This can be addressed by switching cell lines or assessing the expression status of tagged bait proteins. The expression levels of endogenous proteins in various cell lines can be referenced from databases such as Proteomic DB or the Human Protein Atlas.
b. Low affinity purification capability of the IP antibody or failure of the WB antibody to recognize the bait protein. It is recommended to test different antibodies, with reference to validated antibody databases (https://www.labome.com/index.html).
Excessive bands in the input lane indicate poor specificity of the IP antibody, leading to false-positive results. The more non-specific bands other than the bait protein appear, the more false-positive results are introduced. It is generally advisable to replace the IP antibody with one that has better specificity for the IP experiment.
Faint Bait Protein Bands in the IP Lane
Typically, after IP affinity purification/enrichment, the concentration of the bait protein increases. Therefore, in an ideal IP-WB result, the bait protein band in the IP lane should be darker than that in the input lane.
If the bait protein band in the IP lane is lighter than that in the input lane, it indicates poor enrichment of the bait protein during the IP process, often due to improper experimental procedures, insufficient amounts of IP antibody, or low affinity of the IP antibody.
If the bands of antibody light and heavy chains stain very dark, indicating sufficient IP antibody usage, yet the bait protein band is still lighter than that in the input lane, it is usually advisable to replace the antibody with one of better efficacy.
Significant Differences Between Control and IP Lanes
The intensity of antibody light and heavy chain bands can be used to assess the amount of IP antibody used. Ideally, the experimental and control groups should use the same amount of antibody. However, if the experimental design employs blank control IgG from the same species as the experimental antibody, ensuring identical antibody concentrations may be challenging. If there is a significant difference between the lanes of the experimental and control groups, it may indicate issues with the specific settings of the experimental and control groups, necessitating adjustments to antibody amounts, antibody types, or other experimental parameters.
Summary
In the results of an IP-WB experiment, three main aspects are typically focused on:
1. Protein bands in the input lane can be used to assess the specificity of the IP antibody and determine the location of the bait protein band.
2. Differences in bait protein bands between the IP and IgG/control lanes are used to determine whether the bait protein has been successfully purified and enriched through the IP experiment.
3. Differences in bait protein bands among the input, output, and flowthrough lanes in the experimental group can be used to assess the efficiency of bait protein purification and enrichment in the IP experiment.
It should be noted that the IP-WB described here refers to the WB experiment targeting the bait protein after the IP experiment, typically serving as a pre-experiment for IP-MS to evaluate the success of the IP experiment.
It is important to note that while protein quantification is often performed based on band intensity in WB experiments, the quantitative performance of WB staining is limited, with non-ideal linear range and accuracy. Even with the same samples and procedures, adjusting parameters such as WB staining reagent dosage and exposure time can greatly affect the relative intensity of bands. The intensity of WB staining bands cannot be used to determine absolute quantitative information of the corresponding proteins; deeper staining does not necessarily indicate higher protein content.
Moreover, due to differences in performance, principles, etc., between WB and mass spectrometry (MS) detection, IP-WB experiment results cannot fully indicate IP-MS experiment results. A successful IP-WB experiment does not guarantee ideal IP-MS experiment results, and suboptimal IP-WB results may still yield better results through IP-MS.
In conclusion, while IP-WB serves as an important preliminary experiment for IP-MS, its results should be interpreted cautiously, and further validation through complementary techniques such as IP-MS is often necessary.
Reference
- Oelmann, Elisabeth, et al. "Expression of interleukin-1 and interleukin-1 receptors type 1 and type 2 in Hodgkin lymphoma." PloS one 10.9 (2015): e0138747.
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