Immunoprecipitation (IP) combined with Western Blot (WB) is a crucial 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.

The Experimental Process of IP-WB

The classic IP-WB experiment involves several main steps: extracting proteins from cells or tissues using a mild lysis buffer, adding antibodies and beads for incubation, centrifugation or magnetic separation to remove free proteins, eluting proteins from the beads, separating protein components by SDS-PAGE, transferring to a membrane, and incubating with primary and secondary antibodies. The target protein signal is visualized in the WB results through secondary antibody-mediated imaging.

Specific experimental steps include cell lysis, antibody incubation, washing, elution, electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and imaging. Antibodies used in step B of the IP experiment and primary antibodies in step H of the WB experiment are crucial factors influencing IP-WB results. The former targets the bait protein, while the latter can target either the bait protein or its interacting proteins. Successful purification of the bait protein in the IP experiment is often a prerequisite for verifying interactions.

Ideal Analysis of IP-WB Results

In an IP-WB experiment, using whole-cell lysate as a positive control and a blank IP as a negative control is standard practice. An ideal IP-WB experiment should include at least three lanes of WB imaging: input, IP, and IgG/control. Additional negative controls for WB detection can enhance IP result evaluation.

An ideal IP-WB experiment typically shows:

Input lane: Represents whole-cell lysate before IP incubation. Bands of the bait protein without non-specific bands indicate normal expression and high antibody specificity.

IP lane: Contains the protein sample after antibody incubation, washing, and elution. Strong bait protein bands suggest successful enrichment and purification.

IgG/control lane: Shows the blank control group sample with no bait protein bands.

Common Issues in IP-WB Results

Issues in IP-WB results include:

Appearance of bait protein bands in control lane: Possible reasons include indistinguishability from antibody chains or cross-contamination. Solutions involve using different antibodies or investigating experimental design.

Absence or excessive bands in the input lane: Reasons include low expression of the bait protein or low affinity of antibodies. Solutions include switching cell lines or testing different antibodies.

Faint bait protein bands in the IP lane: Indicates poor enrichment due to experimental errors or low antibody affinity. Solutions involve adjusting experimental procedures or using more effective antibodies.

Significant differences between control and IP lanes: Indicates issues with experimental settings, requiring adjustments to antibody amounts or types.

Summary

In IP-WB experiments, focus is on protein bands in the input lane, differences between IP and control lanes, and bait protein bands among different lanes. IP-WB serves as a preliminary experiment for IP-MS, but results should be interpreted cautiously. Further validation through complementary techniques like 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.