AI Article Synopsis
- Current methods like pull-downs and immunoprecipitations for studying protein interactions can suffer from nonspecific binding, leading to false positives in results.
- The proposed method combines stable isotope labeling in cell culture (SILAC) with affinity purification and quantitative tandem mass spectrometry to improve the accuracy of protein interaction analysis.
- This approach not only helps eliminate contaminants but also enables the comparison of interaction patterns between different protein variants and examines changes in protein complexes due to varying activity states.
Article Abstract
Pull-downs based on tag fusion proteins as well as immunoprecipitations (IP) are widely used methods to analyze protein interactions. Selectivity and specificity of both methods are compromised by nonspecific binding to the capture agent or carrier beads thereby generating false positives. Here, we provide a method combining stable isotope labeling of amino acids in cell culture (SILAC) with affinity purification, coupled to quantitative tandem mass spectrometry. It permits the analysis of protein interactions with high sensitivity, while being able to discriminate contaminants and nonspecific binders. Besides pruning out contaminants, high-resolution MS data combined with quantitative proteomics software allow the comparative analysis of protein interaction patterns of different protein variants, for example mutated versus normal protein variant or of regulatory changes in a given protein complex due to different states of activity.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-4939-1142-4_13 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!