Apr 30, 2025

Public workspaceNU Auxin Induced protein degradation

This protocol is a draft, published without a DOI.
  • De Xing1,
  • Ozlem Neyisci1,
  • tao.bai1,
  • adli1
  • 1Northwestern University
Gina Kirsammer
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Protocol Citation: De Xing, Ozlem Neyisci, tao.bai, adli 2025. NU Auxin Induced protein degradation. protocols.io https://protocols.io/view/nu-auxin-induced-protein-degradation-d7ka9kse
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2025
Last Modified: April 30, 2025
Protocol Integer ID: 126306
Funders Acknowledgements:
Molecular and Cellular Characterization of Essential human Genes
Grant ID: UM1HG012649
Abstract
IAA allows OsTir to recognize and target AID(degron)-tagged proteins for degradation. If IAA is depleted, protein expression will return, as regulated by endogenous mechanisms.

Induction may be performed with either IAA, 5-Ph-IAA, or 5-Ad-IAA. >90% protein depletion is typically observed within an hour.
Materials
1. 3-Indoleacetic acid (IAA): Sigma I2886-5G (*do not use for nanodegron)
OR
5-Ph-IAA: MedChem Express, HY-134653 (similar efficacy to IAA at a lower dose)
OR
5-Ad-IAA: TCI Chemicals A3390-10MG
2. DMSO
3. Cell line expressing OsTir and degron-tagged protein of interest

Before start
IAA is only effective for the full length 68 AA degron
**Degradation for 22 Amino Acid nanodegron-tagged proteions requires 5-Ph-IAA (synthetic)
Induced degradation of degron-tagged proteins in IPSC
Induced degradation of degron-tagged proteins in IPSC
Prepare induction agent stock, either IAA or 5-Ph-IAA
a. 3-Indoleacetic acid (IAA) (Sigma I2886-5G) *Effective for full length 68 AA degron only.
Prepare as a 1 M stock in DMSO
b. 5-Ph-IAA (MedChem Express, HY-134653):5-Ph-IAA is efficient as IAA at a much lower dose.
Prepare as a 1mM stock in DMSO.
c. 5-Ad-IAA (TCI Chemicals A3390-10MG)
Prepare as a 1mM stock in DMSO.
d. Stock may be repared fresh or frozen at -20°C for 1 year or -80°C for 2 years
Treat cells (density: 1 million KOLF2.2J cells per well of 6-well-plate) with 500 uM IAA* or 1uM 5-Ph-IAA*/5-Ad-IAA or DMSO (Control). * Optimal concentration should be determined for each experimental system.
>90% protein suppression is typically observed within 1 hour.
Verify depletion by protein-directed methods (Western blot, IF) with gene-specific or anti-3x-flag antibodies.
Repeat treatment daily to maintain protein loss.