Published in STAR Protocols
Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senesc...
Published in STAR Protocols
Here, we describe a fractionation protocol optimized to quantify changes in relative abundance of the chromatin-bound proteome (chromatome) by tandem mass tag multiplexing-based tandem mass spectrometry. It has been applied to yeast cells before and after exposure to DNA damaging drugs to characterize changes in chromatin composition induced by the...
Published in STAR Protocols
We present a comprehensive and robust protocol to track the dynamics of all proteins in a complex in yeast cells. A single member of the protein assembly is tagged and conditionally expressed minimizing the perturbations to the protein complex. Then, SILAC labeling and affinity purification are used for the assessment of the whole protein complex d...
Published in STAR Protocols
Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze prot...
Published in STAR Protocols
This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2. Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET substrates. The protocol is based on an aminomethyl coumarin substrat...
Published in STAR Protocols
Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation ...
Published in STAR Protocols
Typical enzymatic inhibition assays often demonstrate improved potency for kinase covalent inhibitors compared to reversible inhibitors. This can primarily be attributed to the irreversible mode of action and could affect the evaluations of the ATP-competitive nature of covalent inhibitors, hindering optimization of these compounds. Here, we descri...
Published in STAR Protocols
Membrane-bound cargos in cells are generally transported by multiple kinesin motors. Quantifying the bimolecular on-rate of motors for their microtubule track is important for understanding of multi-motor transport but is complicated by diffusion of the motors in the plane of the lipid bilayer. Here, we describe a method to measure the kinesin on-r...
Published in STAR Protocols
We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein ...
Published in STAR Protocols
Identifying drugs targeting p53 remains a major focus of precision oncology, with over twenty compounds that can rescue p53 mutants reported. Here, we suggest three easily accessible assays to determine the thermostability, protein folding, and transcriptional activity of p53 mutants—the go-to criteria for evaluating a rescue compound that acts by ...
