What is cycloheximide chase assay?
Cycloheximide 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 synthesis.
How do you use cycloheximide?
Directions for Use: Cycloheximide is supplied as a lyophilized powder. For a 10 mg/ml stock, carefully weigh out and reconstitute 50 mg in 5 ml DMSO or EtOH. Working concentrations and length of treatments vary depending on the desired effect, but it is typically used at 5-50 µg/ml for 4-24 hours.
Does cycloheximide expire?
Cycloheximide is also soluble in most organic solvents, including ethanol, with the exception of saturated hydrocarbons. Store desiccated at 2-8 °C. The product as supplied should be stable for 5 years if properly stored.
How does cycloheximide inhibit protein synthesis?
Cycloheximide is most commonly used for this purpose in biological research. It blocks protein synthesis through interfering with the translocation step (movement of two tRNA molecules and mRNA in relation to the ribosome) and thus blocking translation elongation (61).
How do you make a cycloheximide solution?
Procedure
- Remove the cycloheximide from cold storage (4°C) and allow to equilibrate to room temperature for a few minutes to minimize moisture uptake.
- Weigh the cycloheximide in the fume hood.
- Add the ethanol and mix to dissolve.
- Store in a foil covered, sealed bottle in cold storage (4°C).
How long can you treat cells with cycloheximide?
4-24 hours
Working concentrations and length of treatments vary depending on the desired effect, but it is typically used at 5-50 µg/ml for 4-24 hours.
What does cycloheximide do to cells?
Cycloheximide is a protein synthesis inhibitor in eukaryotes. Although its precise mechanism of action has yet to be fully elucidated, it has been shown to inhibit translation elongation through binding to the E-site of the 60S ribosomal unit and interfering with deacetylated tRNA (1-3).
What is cycloheximide soluble in?
Cycloheximide is soluble in organic solvents such as ethanol and DMSO, which should be purged with an inert gas. It is also soluble in water. The solubility of cycloheximide in ethanol and DMSO is approximately 25 mg/ml and approximately 20 mg/ml in water.
How do you store cycloheximide powder?
What cellular process does cycloheximide inhibit?
Among the known inhibitors of eukaryotic translation is cycloheximide (CHX, 1), the most common laboratory reagent used to inhibit protein synthesis (Fig. 1). CHX has been shown to block the elongation phase of eukaryotic translation. It binds the ribosome and inhibits eEF2-mediated translocation2.
What is the action of cycloheximide?
Cycloheximide is a naturally occurring fungicide produced by the bacterium Streptomyces griseus. Cycloheximide exerts its effects by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in relation to the ribosome), thus blocking eukaryotic translational elongation.
How do you dilute cycloheximide?
Preparation Instructions Cycloheximide has been reported to be soluble up to 2% (w/v) in water (20 mg/ml). dissolve slowly in water and this process may be aided by mixing or sonication. More dilute solutions (5mg/ml) may be prepared without sonication.