Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE

被引:5
作者
Bruechert, Stefan [1 ]
Joest, Eike F. [1 ]
Gatterdam, Karl [1 ,2 ]
Tampe, Robert [1 ]
机构
[1] Goethe Univ Frankfurt, Bioctr, Inst Biochem, Max von Laue Str 9, D-60438 Frankfurt, Germany
[2] Univ Bonn, Biomed Ctr, Inst Biol Struct, Venusberg Campus 1, D-53127 Bonn, Germany
关键词
ELECTROPHORETIC TRANSFER; POLYACRYLAMIDE-GELS; PROTEINS; NITROCELLULOSE; NTA;
D O I
10.1038/s42003-020-0852-1
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页数:7
相关论文
共 21 条
[1]   Structure of the human MHC-I peptide-loading complex [J].
Blees, Andreas ;
Januliene, Dovile ;
Hofmann, Tommy ;
Koller, Nicole ;
Schmidt, Carla ;
Trowitzsch, Simon ;
Moeller, Arne ;
Tampe, Robert .
NATURE, 2017, 551 (7681) :525-+
[2]  
BURNETTE WN, 1981, ANAL BIOCHEM, V112, P195, DOI 10.1016/0003-2697(81)90281-5
[3]   The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His-tagged Proteins in vitro and in Cells [J].
Gatterdam, Karl ;
Joest, Eike F. ;
Gatterdam, Volker ;
Tampe, Robert .
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2018, 57 (38) :12395-12399
[4]   Super-Chelators for Advanced Protein Labeling in Living Cells [J].
Gatterdam, Karl ;
Joest, Eike F. ;
Dietz, Marina S. ;
Heilemann, Mike ;
Tampe, Robert .
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2018, 57 (20) :5620-5625
[5]   Reversible site-selective labeling of membrane proteins in live cells [J].
Guignet, EG ;
Hovius, R ;
Vogel, H .
NATURE BIOTECHNOLOGY, 2004, 22 (04) :440-444
[6]   NEW METAL CHELATE ADSORBENT SELECTIVE FOR PROTEINS AND PEPTIDES CONTAINING NEIGHBORING HISTIDINE-RESIDUES [J].
HOCHULI, E ;
DOBELI, H ;
SCHACHER, A .
JOURNAL OF CHROMATOGRAPHY, 1987, 411 :177-184
[7]   Conformation space of a heterodimeric ABC exporter under turnover conditions [J].
Hofmann, Susanne ;
Januliene, Dovile ;
Mehdipour, Ahmad R. ;
Thomas, Christoph ;
Stefan, Erich ;
Bruechert, Stefan ;
Kuhn, Benedikt T. ;
Geertsma, Eric R. ;
Hummer, Gerhard ;
Tampe, Robert ;
Moeller, Arne .
NATURE, 2019, 571 (7766) :580-+
[8]   Site-specific incorporation of fluorescent probes into protein:: Hexahistidine-tag-mediated fluorescent labeling with (Ni2+:Nitrilotriacetic acid)n-fluorochrome conjugates [J].
Kapanidis, AN ;
Ebright, YW ;
Ebright, RH .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2001, 123 (48) :12123-12125
[9]   Modulation of protein properties in living cells using nanobodies [J].
Kirchhofer, Axel ;
Helma, Jonas ;
Schmidthals, Katrin ;
Frauer, Carina ;
Cui, Sheng ;
Karcher, Annette ;
Pellis, Mireille ;
Muyldermans, Serge ;
Casas-Delucchi, Corella S. ;
Cardoso, M. Cristina ;
Leonhardt, Heinrich ;
Hopfner, Karl-Peter ;
Rothbauer, Ulrich .
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2010, 17 (01) :133-U162
[10]   Live-cell protein labelling with nanometre precision by cell squeezing [J].
Kollmannsperger, Alina ;
Sharei, Armon ;
Raulf, Anika ;
Heilemann, Mike ;
Langer, Robert ;
Jensen, Klavs F. ;
Wieneke, Ralph ;
Tampe, Robert .
NATURE COMMUNICATIONS, 2016, 7