Cycloheximide-chase assays
WebApr 30, 2024 · Cycloheximide is de-novo protein synthesis inhibitor that acts by inhibit translation elongation through binding to the E-site of the 60S ribosomal unit. This, in turn, arrests protein synthesis allowing investigators to follow degradation profile of proteins without having to worry about the effects of ongoing protein synthesis. WebCycloheximide chase assays are an experimental technique used in molecular and cellular biology to measure steady state protein stability. Cycloheximide is a drug that inhibits the elongation step in eukaryotic protein translation, thereby preventing protein …
Cycloheximide-chase assays
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WebApr 7, 2024 · The stability of AUF1 and its degradation by ubiquitin-proteasome pathway were examined by cycloheximide chase analysis and co-immunoprecipitation assay. The regulation of AUF1 on nuclear factor E2-related factor 2 (NRF2) and activation transcription factor 3 (ATF3) was explored by RNA immunoprecipitation (RIP), RNA pull-down, and … WebTo measure protein half-lives in cultured mammalian cells (HEK293), we used cycloheximide at a concentration of 100 mkg/ml. The control protein which is GFP …
WebMay 28, 2024 · Cycloheximide chase assay The protein degradation rate of GPX4 was tested in HL-60 cells after treatment with cyclophosphamide (10 μM) in the absence or presence of cycloheximide (10 μM) for 1–4 h. Whole cell lysates were analyzed for GPX4 and ACTB by Western blot analyses. WebApr 1, 2024 · Herein, we report the development of a novel cycloheximide (CHX)-chase degradation assay we termed SNAP-GPCR-CHX. This assay leverages the self-labeling SNAP-epitope reporter tag to facilitate the monitoring of SNAP-tagged GPCR protein expression levels using LICOR near-infrared (NIR) imaging–polyacrylamide gel …
WebAug 26, 2024 · Cycloheximide Chase Assay SpCENP-A NT mutant overexpression strains were pre-inoculated and incubated in EMM-Leu media supplemented with 100 μM of thiamine overnight at 30 °C with shaking. The induction of exogenous protein expression in log-phase strains bearing CENP-A N -terminal truncation plasmid constructs was … WebApr 18, 2016 · The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. …
WebCycloheximide chase assay of HA-MARCH2. (A) HEK293 cells were transfected with 1µg HA-MARCH2. After 24 h, 100µg/ml cycloheximide (CHX) was added to the culture medium and cell lysates were...
WebNov 18, 2024 · Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. potplayer configurarWebFeb 5, 2024 · Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. toucher collerWebJan 5, 2015 · It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full … toucher connectWebWe show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins. protein degradation protein stability protein turnover ubiquitin ligase fusion protein E3 ubiquitin ligase haloalkane haloalkane hydrolase HaloTag potplayer command line argumentsWebLuciferase assays were carried out in 96-well white solid bottom plates. d -Luciferin (50 μl of 1 mg/ml in PBS) was added into each well containing 50 μl of medium and incubated at room temperature with shaking at 500 … toucher conjugationWebCycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we ... toucher conjugaisonWebCycloheximide is known to inhibit protein synthesis of symbiotic Chlorella of the ciliate P. bursaria preferentially, but it only slightly inhibits host protein synthesis (Ayala and Weis, … potplayer convert mp4 to mp3