Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture
| Autor: | David M. Suter, Andrea B. Alber |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
education.field_of_study Lysis General Immunology and Microbiology Chemistry General Chemical Engineering General Neuroscience Population Cell Culture Techniques Protein degradation Fusion protein General Biochemistry Genetics and Molecular Biology SNAP-tag Mice 03 medical and health sciences 030104 developmental biology Microscopy Fluorescence Cell culture Live cell imaging Biophysics Fluorescence microscope Animals Humans education Developmental Biology |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/56604-v |
| Popis: | Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined. |
| Databáze: | OpenAIRE |
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