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- Title
Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE.
- Authors
Brüchert, Stefan; Joest, Eike F.; Gatterdam, Karl; Tampé, Robert
- Abstract
Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles. Brüchert et al. describe the application of high-affinity super-chelator probes that are linked to a fluorophore for the detection of His-tagged proteins using PAGE. Their system has the advantage over conventional antibody staining in the ease of application and ultra-sensitive detection.
- Subjects
NUCLEOPROTEINS; NITRILOTRIACETIC acid; FLUOROPHORES; MEMBRANE proteins; CHROMATOGRAPHIC analysis
- Publication
Communications Biology, 2020, Vol 3, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-020-0852-1