Formedium products mentioned in Tuning Degradation to Achieve Specific and Effecient Protein Depletion
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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|Β-estradiol||Sigma Aldrich||E2758-1G||10 mM solution in ethanol. Store at -20 °C|
|DMSO||Alfa Aesar||42780||DMSO should be solid at 4 °C|
|IAA 1H-Indole-3-acetic acid||Across Orgainics||122150100||Auxin 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.|
|Methanol||Fisher Scientific||M/4000/PC17||CAUTION Toxic and flammable|
|Phusion High-Fidelity DNA Polymerase||NEB||M0530|
|SCSM single drop-out –ura||Formedium||DSCS101|
|Yeast nitrogen base without amino acids with amonium sulphate||Formedium||CYN0410|
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  and by Wellcome funding  to JD Beggs. Work in the Wellcome Centre for Cell Biology is supported by Wellcome core funding .
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).