Biopharma Insights We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.
Model organism
Chlamydomonas reinhardtii
Steps
1
Lyse cells and crosslink proteins
Perform cell lysis and chemical crosslinking to stabilize protein-protein interactions for subsequent analysis.
▶ 02:30
2
Immunoprecipitate target protein complexes
Use antibodies to pull down the protein of interest along with its interaction partners, with or without ATP to modulate affinities.
▶ 05:22
3
Prepare samples for nano-LC-MS/MS
Process immunoprecipitated samples, including digestion and cleanup, for subsequent quantitative mass spectrometry analysis.
▶ 07:06
4
Analyze results for HSP70B interactors
Review representative results showing identification of HSP70B interactions with its nucleotide exchange factor CGE1.
▶ 10:50