Development of Diubiquitin-Based FRET Probes to Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes
Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner.
Geurink, Paul P.,Van Tol, Bianca D. M.,Van Dalen, Duco,Brundel, Paul J. G.,Mevissen, Tycho E. T.,Pruneda, Jonathan N.,Elliott, Paul R.,Van Tilburg, Gabri?lle B. A.,Komander, David,Ovaa, Huib
NEW TECHNOLOGY TO CONJUGATE THE TACCALONOLIDE MICROTUBULE STABILIZERS WITH LINKERS/PAYLOADS
The present disclosure is concerned with taccalonolide analogs and conjugated taccalonolide analogs useful as cellular probes and in the treatment of, for example, hyperproliferative disorders such as cardiovascular diseases and cancer. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.
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Paragraph 0486; 0487
(2020/09/27)
Site-specific, reversible and fluorescent immobilization of proteins on CrAsH-modified surfaces for microarray analytics
A novel technique for protein immobilization onto CrAsH-modified surfaces is presented. This approach enables an efficient, reversible and fluorogenic immobilization of proteins. Moreover, expressed proteins can also be directly immobilized from cellular lysates without prior purification. The immobilized proteins are suitable for protein-protein interaction studies and the fluorescence enhancement upon immobilization can be employed for the direct detection of the immobilized protein without the need for secondary detection methods. This journal is
Separating the isomers - Efficient synthesis of the N-hydroxysuccinimide esters of 5 and 6-carboxyfluorescein diacetate and 5 and 6-carboxyrhodamine B
Diacetate protection of 5 and 6-carboxyfluorescein followed by synthesis of the N-hydroxysuccinimide esters allowed ready separation of the two isomers on a multi-gram scale. The 5 and 6-carboxyrhodamine B N-hydroxysuccinimide esters were also readily synthesised and separated.
Brunet, Aurlie,Aslam, Tashfeen,Bradley, Mark
p. 3186 - 3188
(2015/02/05)
FLUORESCENT SUBSTRATE FOR DETECTION OF ENZYMATIC ACTIVITY OF NITRILE-RELATED ENZYME
The object of the present invention is to provide a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme. The present invention provides a compound represented by formula (I) and a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme, which comprises the compound.
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Paragraph 0083; 0084
(2013/03/28)
Cell-permeable small molecule probes for site-specific labeling of proteins.
We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.
Yeo, Dawn S Y,Srinivasan, Rajavel,Uttamchandani, Mahesh,Chen, Grace Y J,Zhu, Qing,Yao, Shao Q
p. 2870 - 2871
(2007/10/03)
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