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  • Auxin Proteo ControllerAuxin Inducible Degron System : AID System

Unique and powerful new tool which has applied auxin-inducible degradation of target protein to animal cells and yeast.

Auxin Proteo Controller
Auxin Inducible Degron System : AID System


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ico_caution_s.pngContents of Anti-AID Tag have been changed. The AID kit is containing Anti-AID Tag (IAA17) Protein Mouse Monoclonal Antibody, not Rabbit Polyclonal.

Background

This product "AID System" can control protein level of interest in a reversible fashion in cultured cells and can directly induce degradation of target protein, therefore, protein depletion can be achieved..
Moreover, "AID System" can induce degradation much faster (typically in 30 minutes) than other systems controlling promoter activity and allows to transplant a plant specific degradation pathway involved in the auxin response to animal cells.


AID system flyer download [PDF]

Application

™ Protein level is directly controlled by the addition of auxin which induces a proteasome-dependent degradation of target protein.
™ A 25-kD degron can be attached to either the N or C terminus of target protein.
™ Fast control of protein level.
™ Both nuclear and cytoplasmic proteins can be controlled.
™ Very low toxicity of auxin to animal cells.
™ Easy system construction by incorporation of just one vector.

Principle

The vector plasmid used in this system allows expression of two proteins, the F-box TIR1 protein and the protein of interest fused to a 25-kD degron which confer inducible degradation to the target protein. The genes encoding those two proteins are transcribed as a bicistronic mRNA which makes the two proteins simultaneously by using an internal ribosome binding site (IRES).
TIR1 forms an auxin-dependent E3 ubiquitin ligase complex, SCFTIR1, by utilizing endogenous SCF subunits in cells.

In the presence of auxin, SCFTIR1 recognizes and works as an ubiquitin ligase to polyubiquitilate the degron fused to the target and then the fusion protein is immediately degraded by the proteasome.
If auxin removed, the fusion protein can be re-expressed typically within 1-2 hours.

Basic specification

< Plasmid Structure >

• pAID1.1-N Vector

pAID1.1-C Vector

 

< Rapid Degradation of Target Protein >

Depletion of a GFP reporter fused to aid (GFP-aid) after addition of auxin A 293 stable line expressing GFP-aid was established using pAID1.1-C. Cells were taken at indicated time points for immunoblotting after addition of 500 μM IAA (a natural auxin) to culture medium. The most of GFP-aid was depleted within 30 minutes in the presence of IAA.

< Free Expression Control >

Modulation of expression level using different auxin concentrations.
A stable 233 line as shown above was treated with different concentrations of auxin. Expression levels of GFP-aid were inversely correlated with auxin concentrations.

< Reversibility of Switch On/OFF of Expression >

After degradation of GFP-aid in 233 cells, the cells were transferred to culture medium without auxin in order to see re-expression of GFP-aid. As shown here, GFP-aid was re-expressed within 1 hour after removal of auxin.

Experiment example

< Characterization of Auxin Proteo Controller “AID System” >

In order to control protein level conditionally, the Tet On/OFF systems have been wildly used with which expression ca be controlled at the level of transcription. Therefore, depletion of target protein depends on its half-life. As target protein is directly degraded in the presence of auxin,protein depletion can be achieved much faster in the APC system. Using chicken DT40 cell line, a kinetochore CENP-H protein were controlled either by APC or by the Tet OFF system. After depletion treatments, cells were stained to see CENP-H (green) and nuclear DNA (blue) at the indicated time points. CENP-H was depleted within 30 minutes by APC. On the other hand, CENP-H was detected up to 24 hours when controlled using Tet OFF.

Contents

AID System consists of two types of plasmid and Auxin (IAA).
One plasmid is pAID1.1-N Vector which is combined with N-terminal of target protein.
The other one is pAID1.1-C Vector which is combined with C-terminal of target protein.
IAA is Auxin which induces degradation of target aid protein.

FQA

FQA about AID System

For consumers who order AID vectors
Agreement for AID System Usage

Please accept the following terms:

1. We will not use the AID System for testing in humans.
2. We will not replicate any AID System vector without written permission from BioROIS.
3. We shall not include any AID System component or derivative thereof in any patent application without expressed written permission from BioROIS.
4. We shall not transfer, deliver, sell, assigh or let use any AID System component, information or derivative to any persons or to any organization beyond the scope of our control or responsibility without prioi written consent from BioROIS.
5. We shall inform BioROIS of any data published using AID System components or derivatives. 

Please download and print the agreement form (PDF file). Please fill out and fax to Cosmo Bio Co., Ltd. before placing your order.

Download the form
References

An auxin-based degron system for the rapid depletion of proteins in nonplant cells
NATURE METHODS VOL.6 NO.12 DECEMBER 2009 P917 - P923


This product is cited in following article
Sakurai A et al., Activated Ras protein accelerates cell cycle progression to perturb Madin-Darby canine kidney cystogenesis, J Biol Chem., 287(38):31703-11, 2012

Product List


Product Name Cat# Quantity Price

pAID1.1-N Vector DataSheet ,

BRS-APC001A

20 UG N/A

 


Product Name Cat# Quantity Price

pAID1.1-C Vector DataSheet ,

BRS-APC002A

20 UG N/A


Product Name Cat# Quantity Price

IAA(α) ,

BRS-APC003A

1 ML N/A


Product Name Cat# Quantity Price

Anti AID Tag (Mouse)  DataSheet

BRS-APC004AM

100 UL N/A


Product Name Cat# Quantity Price

pAID1.1-N Vector and IAA(α) DataSheet ,

BRS-APC005A

1 SET N/A


Product Name Cat# Quantity Price

pAID1.1-C Vector and IAA(α) DataSheet ,

BRS-APC006A

1 SET N/A


Product Name Cat# Quantity Price

pAID1.1-N Vector and Anti-AID Tag Mouse Monoclonal DataSheet ,

BRS-APC007A

1 SET N/A


Product Name Cat# Quantity Price

pAID1.1-C Vector and Anti-AID Tag Mouse Monoclonal DataSheet ,

BRS-APC008A

1 SET N/A


Product Name Cat# Quantity Price

pAID1.1-N Vector, IAA(α) and Anti-AID Tag Mouse Monoclonal DataSheet ,

BRS-APC009A

1 SET N/A


Product Name Cat# Quantity Price

pAID1.1-C Vector, IAA(α) and Anti-AID Tag Mouse Monoclonal DataSheet ,

BRS-APC010A

1 SET N/A


Product Name Cat# Quantity Price

Auxin-Inducible Degron (AID) System Total Set DataSheet ,

BRS-APC011A

1 SET N/A

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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