What are the best practices for measuring protein-protein interactions?

1 Choose the right method

Different methods for measuring PPIs have their own advantages and limitations, such as sensitivity, specificity, throughput, scalability, and compatibility with different samples and conditions. For example, co-immunoprecipitation (Co-IP) uses antibodies to isolate and identify proteins that interact with a target protein in a cell lysate or a tissue sample. Affinity purification-mass spectrometry (AP-MS) combines affinity purification of a target protein or a protein complex with mass spectrometry to identify and quantify the interacting proteins. Yeast two-hybrid (Y2H) uses a reporter gene expression system in yeast to detect the interaction between two proteins that are fused to different domains of a transcription factor. Fluorescence resonance energy transfer (FRET) uses fluorescent probes that emit or absorb light at different wavelengths to measure the distance and interaction between two proteins in living cells. Bioluminescence resonance energy transfer (BRET) is similar to FRET, but uses bioluminescent probes that generate light without external excitation to measure the interaction between two proteins in living cells. Thus, it is important to consider which method best suits your research question, experimental design, and available resources when selecting the appropriate method for measuring PPIs.

2 Optimize the experimental conditions

Once you have chosen the method for measuring PPIs, you should optimize the experimental conditions to ensure the quality and reproducibility of your results. Factors to consider include protein expression and purification, using appropriate expression systems and buffers to produce and purify proteins with high yield and purity. Additionally, you should use suitable labels and detection methods for your proteins, depending on the method you use. It's also important to consider protein concentration and ratio, buffer composition and pH, as well as temperature and time settings in order to mimic physiological or pathological conditions, avoid nonspecific interactions or interference, and minimize the effects of thermal or kinetic fluctuations. Verifying the identity, integrity, and activity of your proteins using techniques such as SDS-PAGE, Western blotting, and enzyme assays is key for obtaining accurate results.

3 Validate and verify the results

After you have measured your PPIs, it is essential to validate and verify your results to confirm the specificity and relevance of them. To do this, you should perform positive and negative controls in your experiments to assess the reliability and accuracy of your method and data. Additionally, repeating your experiments at least three times will ensure the consistency and reproducibility of your results. Moreover, using multiple methods to measure your PPIs can provide complementary and corroborative information. Furthermore, functional assays can demonstrate the biological consequences and implications of your PPIs. All these strategies can help you reduce the biases and errors of each method, as well as evaluate the significance and variation of your results.

4 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?