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Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening

Authors: Gregory B. Craven, David J. Mann and Alan Armstrong, date: 12/20/2020, view: 381, Q&A: 0

[Abstract]

Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of

...

Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag

Author: Junshi Yazaki, date: 09/20/2020, view: 1445, Q&A: 0

[Abstract] Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification ...

Simple Method to Determine Protein Redox State in Arabidopsis thaliana

Authors: Keisuke Yoshida and Toru Hisabori, date: 06/05/2019, view: 3312, Q&A: 0

[Abstract] Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana ...

Detection of Disulfides in Protein Extracts of Arabidopsis thaliana Using Monobromobimane (mBB)

Authors: Shin‐nosuke Hashida and Maki Kawai-Yamada, date: 03/05/2019, view: 3360, Q&A: 0

[Abstract] Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple ...

Preparation of Silk Fibroin Nanoparticles and Enzyme-Entrapped Silk Fibroin Nanoparticles

Author: Yu-Qing Zhang, date: 12/20/2018, view: 4646, Q&A: 0

[Abstract] After silk fiber is degummed in boiling 0.2% Na₂CO₃ solution, the degummed fibroin fiber could be dissolved in highly concentrated neutral salts such as CaCl₂. The partially degraded polypeptides of silk fibroin, commonly called as regenerated liquid silk, could be obtained via water dialysis. The silk fibroin ...

A Method for SUMO Modification of Proteins in vitro

Authors: Christine C. Lee, Bing Li, Hongtao Yu and Michael J. Matunis, date: 10/05/2018, view: 3743, Q&A: 0

[Abstract] The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin ...

Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry

Authors: Bingbing Ren, Ahmed Mahmoud Mohammed Sayed, Hwei Ling Tan, Yu Keung Mok and Ee Sin Chen, date: 09/20/2018, view: 4341, Q&A: 0

[Abstract] Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the ...

Characterization of Protein Domain Function via in vitro DNA Shuffling

Authors: Kathy Hiu Laam Po, Edward Wai Chi Chan and Sheng Chen, date: 06/05/2018, view: 3534, Q&A: 0

[Abstract] We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the bla_CTX-M-14 and bla_CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active ...

Histone Deubiquitination Assay in Nicotiana benthamiana

Authors: Shujing Liu and Lars Hennig, date: 03/05/2018, view: 3478, Q&A: 0

[Abstract] Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo ...

In vitro AMPylation Assays Using Purified, Recombinant Proteins

Authors: Matthias C. Truttmann and Hidde L. Ploegh, date: 07/20/2017, view: 4856, Q&A: 0

[Abstract] Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein ...

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