Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE
- 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.
Author: | Stefan Brüchert, Eike F. Joest, Karl GatterdamORCiDGND, Robert TampéORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-531574 |
DOI: | https://doi.org/10.1038/s42003-020-0852-1 |
ISSN: | 2399-3642 |
Parent Title (German): | Communications biology |
Publisher: | Springer Nature |
Place of publication: | London |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2020/03/20 |
Date of first Publication: | 2020/03/20 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2020/05/13 |
Volume: | 3 |
Issue: | Article number: 138 |
Page Number: | 7 |
HeBIS-PPN: | 465942784 |
Institutes: | Biochemie, Chemie und Pharmazie / Biochemie und Chemie |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - Namensnennung 4.0 |