Abstract
The half-life of a particular protein is highly variable, reaching from minutes to hours, over days and weeks to years or even a whole life time of an organism (e.g., α-crystalline of the mammalian eye). Thus, controlling protein activity by proteolysis is the most dramatic and unambiguous decision by any organism, because reconstitution of the destroyed protein activity requires an “expensive” new synthesis. To distinguish degradation from protein synthesis and accumulation only one method comes into consideration—pulse-chase labeling. In our hands, the most accurate method to track the fate of a single protein is radioactive pulse-chase labeling combined with immunoprecipitation. Besides a detailed description of the standard protocol, the general applicability as well as certain improvements of the method will be discussed here.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Reder A, Höper D, Weinberg C, Gerth U, Fraunholz M, Hecker M (2008) The Spx paralogue MgsR (YqgZ) controls a subregulon within the general stress response of Bacillus subtilis. Mol Microbiol 69(5):1104–1120
Reder A, Pöther DC, Gerth U, Hecker M (2012) The modulator of the general stress response, MgsR, of Bacillus subtilis is subject to multiple and complex control mechanisms. Environ Microbiol 14(10):2838–2850
Nakano MM, Lin A, Zuber CS, Newberry KJ, Brennan RG, Zuber P (2010) Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS One 5(1):e8664
Nakano S, Nakano MM, Zhang Y, Leelakriangsak M, Zuber P (2003) A regulatory protein that interferes with activator-stimulated transcription in bacteria. Proc Natl Acad Sci U S A 100(7):4233–4238
Lin AA, Walthers D, Zuber P (2013) Residue substitutions near the redox center of Bacillus subtilis Spx affect RNA polymerase interaction, redox control, and Spx-DNA contact at a conserved cis-acting element. J Bacteriol 195(17):3967–3978
Rochat T, Nicolas P, Delumeau O, Rabatinova A, Korelusova J, Leduc A, Bessieres P, Dervyn E, Krasny L, Noirot P (2012) Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis. Nucleic Acids Res 40(19):9571–9583
Stülke J, Hanschke R, Hecker M (1993) Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol 139(9):2041–2045
Gerth U, Kock H, Kusters I, Michalik S, Switzer RL, Hecker M (2008) Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis. J Bacteriol 190(1):321–331
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Reder, A., Michalik, S., Gerth, U. (2018). How to Assess Protein Stability: Half-Life Determination of a Regulatory Protein in Bacillus subtilis. In: Becher, D. (eds) Microbial Proteomics. Methods in Molecular Biology, vol 1841. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8695-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8695-8_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8693-4
Online ISBN: 978-1-4939-8695-8
eBook Packages: Springer Protocols