Journal
COMMUNICATIONS BIOLOGY
Volume 3, Issue 1, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s42003-020-0852-1
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Funding
- German Research Foundation [GRK 1986, SFB 902, SFB 807]
- Volkswagen Foundation [Az. 91 067]
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Bruchert 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. 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.
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