For Research Use Only. Not for Clinical Use.

Creative Biolabs is one of the world's leading biotechnology companies. With a professional team and years of experience, we have developed a series of innovative drug half-life analysis platforms that can provide you with personalized analysis services.

Cycloheximide

Cycloheximide (CHX), also known as Actidione, is a glutamine antibiotic isolated and identified in 1948 by the German chemist Jared H.Ford. It was first identified as a compound with anti-fungal properties produced by the gram-positive bacterium Streptomyces griseus, which is a cell permeable molecule that specifically inhibits eukaryotic cytosolic translation by impairing ribosomal translocation. At present, cycloheximide has been widely used in the field of biotechnology research, especially popular in research on apoptosis mechanism, protein expression, cell cycle research, pathological model design, etc.

Cycloheximide Chase

Cycloheximide Chase is one of the most classical approaches and almost the first to be utilized for monitoring protein degradation and measuring protein’s half-life in cultured cells. Protein synthesis is inhibited by cycloheximide and the decay of a target protein over time is determined by SDS-PAGE and immunoblot analysis. The generated degradation profiles or curves can be normalized through monitoring, and in parallel to, the abundance of a control stable endogenous protein (for instance, tubulin, GAPDH, actin). This stable control or reference protein should ideally be expressed at roughly equal levels under various physiological conditions and within diverse genetic systems. This method has been applied in different mammalian cell lines to study protein degradation.

Fig.1 Cycloheximide-Chase analysis (Eldeeb, 2019)

Features of CHX-chase

• Growth-based reporter assays reflect steady-state protein levels over an extended period but do not discriminate between the influences of synthesis and degradation. Instead, CHX-chase analysis proteins at a single time point thus can provide a snapshot of steady state protein abundance.
• Compared with other analytical methods, CHX-chase assay is relatively simple to quantify the degradation of a given protein fragment following the addition and continued incubation ('chase') with the protein translation inhibitor.
• The CHX-chase procedure is insensitive to highly denaturing extraction buffers that allow for maximal recovery of proteins from cells.
• CHX-chase method is ill-suited for long-lived proteins owing to the deleterious effects of prolonged global mitigation of protein translation process on the global cellular physiology and function. However, if crucial elements of the degradation machinery are themselves short-lived, their substrates may ultimately be stabilized by the administration of CHX.

Creative Biolabs is dedicated to providing the first-in-class drug half-life assay service for our customers worldwide. If you have any questions about half-life assay by Cycloheximide Chase, please do not hesitate to contact us for more detailed information.

Reference

1. Eldeeb, M. A.; et al. A molecular toolbox for studying protein degradation in mammalian cells. Journal of neurochemistry. 2019, 151(4), 520-533.