VEDRØRENDE BLODSTØRKNINGSTEST: SE https://blog.bionyt.dk//Quick-test
Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC) and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants ( those enriched in precipitates from the wild type as specific interaction partners of the target protein)
https://www.ncbi.nlm.nih.gov/pubmed/23051728 (artikel fra 2012)
Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK): Present screening methods for protein-protein interactions (PPIs) rely on the overexpression of artificial fusion proteins, making it difficult to assess in vivo relevance. Here we combine stable isotope labeling with amino acids in cell culture (SILAC), RNA interference (RNAi), coimmunoprecipitation and quantitative mass-spectrometry analysis to detect cellular interaction partners of endogenous proteins in mammalian cells with very high confidence. We used this screen to identify interaction partners of beta-catenin and Cbl.
QUICKstep and GS-TAP: new moves for protein-interaction analysis.
Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK).
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