Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea.
| Autor: | Kern M; Biochemistry III-Regensburg Center for Biochemistry-Institute for Biochemistry, Genetics and Microbiology, University of Regensburg, Regensburg, Germany., Ferreira-Cerca S; Biochemistry III-Regensburg Center for Biochemistry-Institute for Biochemistry, Genetics and Microbiology, University of Regensburg, Regensburg, Germany. sebastien.ferreira-cerca@ur.de. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2533, pp. 229-246. |
| DOI: | 10.1007/978-1-0716-2501-9_14 |
| Abstrakt: | The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea . (© 2022. The Author(s).) |
| Databáze: | MEDLINE |
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