Published: Vol 5, Iss 1, Jan 5, 2015 DOI: 10.21769/BioProtoc.1374 Views: 62230
Reviewed by: Hsin-Yi ChangGuillermo GomezAnonymous reviewer(s)
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis
Koshi Imami and Tomoharu Yasuda
Apr 20, 2019 8908 Views
Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins
Di Feng [...] Bo Gao
Oct 20, 2022 2683 Views
Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging
Kristina Seiler [...] Mario P. Tschan
Jul 20, 2023 1679 Views
Abstract
Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. 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 cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.
Materials and Reagents
Equipment
Software
Procedure
Determine the time course of cycloheximide chase beforehand. There can be great variation among proteins. For an unfamiliar protein, it is recommended to start with a 4-hour interval and chase till 24 h. Cycloheximide may cause cytotoxicity to certain cells if cells are exposed to it over 20-24 h. It has been known that Slug is a labile protein with a T1/2 of about 40 min (Wang et al., 2009). CL1-5 cells stably overexpressing Flag-tagged wtSlug and non-phosphorylatable Slug-4SA were prepared using lentiviral transduction (Kao et al., 2014). Cells were grown in complete medium with 400 μg/ml G418. To compare the turnover of wtSlug and Slug-4SA, these stable cells were subjected to the cycloheximide chase assay.
Quantification
Quantification was achieved by the MetaMorph software in this case. An analysis guide should be referred to for a first-time user. Basically, regions of measurement are selected and threshold is defined. Draw region of interest on top of the western blot bands and quantitate its total grey signal (this can be done in Image J and any other image analysis software). The values of total thresholded areas are measured; a ratio of Flag-Slug to its β-actin is then calculated. The final Slug protein turnover rate at each time point is the percentage of Slug/β-actin at t = 0 of each experimental group. A plot can be created afterwards.
Representative data
Western blots and quantification images have been published in Oncogene (Kao et al., 2014). For details, please refer to Figure 2f at Kao et al. (2014).
Recipes
Acknowledgments
This work was supported by grants from the National Science Council, Taiwan (NSC99-2628-B-006-031-MY3 NSC101-2325-B-006-018, NSC100-2321-B-002-071 and NSC101-2321-B002-068), National Taiwan University, Taiwan (10R71601-2), and National Institute of Health, USA (R01-GM-094231, to AIN). SP Wang is supported by a Human Frontier Science Program long-term fellowship. This protocol was adapted from previous work published in Oncogene (Kao et al., 2014).
References
Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Kao, S., Wang, W., Chen, C., Chang, Y., Wu, Y., Wang, Y., Wang, S., Nesvizhskii, A. I., Chen, Y., Hong, T. and Yang, P. (2015). Analysis of Protein Stability by the Cycloheximide Chase Assay. Bio-protocol 5(1): e1374. DOI: 10.21769/BioProtoc.1374.
Category
Cancer Biology > General technique > Biochemical assays
Biochemistry > Protein > Synthesis
Molecular Biology > Protein > Detection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link