Formedium products mentioned in Tuning Degradation to Achieve Specific and Effecient Protein Depletion

Introduction

Conditional mutations, such as temperature-sensitive mutants, are a powerful tool for the study of essential proteins, allowing cell growth under the permissive condition but causing loss of function under non-permissive conditions. However, cell metabolism can be seriously perturbed by the change in growth conditions required to induce the defect and may also create off-target effects. Several methods have been developed, in which the protein of interest is conditionally sequestered1 or its expression is controlled2,3 by addition of a small molecule. This protocol uses auxin and the auxin-inducible degron (AID) system to efficiently deplete a target protein.

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Materials

NameCompanyCatalog NumberComments
Adenine sulphateFormediumDOC0230
AgarFormediumAGA03
Β-estradiolSigma AldrichE2758-1G10 mM solution in ethanol. Store at -20 °C
DMSOAlfa Aesar42780DMSO should be solid at 4 °C
GlucoseFisher ScientificG/0500/60
IAA 1H-Indole-3-acetic acidAcross Orgainics122150100Auxin analogue. 1.5 M in DMSO. The solution will be a russet colour and darken as time goes on; a deep red solution should be discarded and a new one made. Store at -20 °C.
MethanolFisher ScientificM/4000/PC17CAUTION Toxic and flammable
Phusion High-Fidelity DNA PolymeraseNEBM0530
PeptoneFormediumPEP03
SCSM single drop-out –uraFormediumDSCS101
Yeast ExtractFormediumYEA03
Yeast nitrogen base without amino acids with amonium sulphateFormediumCYN0410

Acknowledgments

Thanks to Jane Reid for initiating this programme, Barbara Terlouw for development, Vahid Aslanzadeh for the “ura looper” constructs and Susana de Lucas for many helpful discussions. This work was supported by a scholarship to GIMO from the Consejo Nacional de Ciencia y Tecnología, Mexico (CONACYT) and the University of Edinburgh School of Biological Sciences, a Wellcome PhD studentship to IEM [105256] and by Wellcome funding [104648] to JD Beggs. Work in the Wellcome Centre for Cell Biology is supported by Wellcome core funding [092076].

Cite this Article

Barrass, J. D., Mendoza-Ochoa, G. I., Maudlin, I. E., Sani, E., Beggs, J. D. Tuning Degradation to Achieve Specific and Efficient Protein Depletion. J. Vis. Exp. (149), e59874, doi:10.3791/59874 (2019).