Auxin-Inducible Degron (AID) System Total Set

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1 Auxin-Inducible Degron (AID) System Total Set This AID System Catalog No. APC011A Can control expression of target protein in a reversible fashion in cultured cells. Can regulate degradation of target protein in a different manner from promoter system. Is an unique and a powerful new tool which has applied auxin-inducible degradation of target protein to animal cells and yeast. Charactoristics Regulation of the amount of protein expression throughout induction of proteolysis system. Highly rapid expression control system in comparing with any other known systems. Rapid Proteolysis induced by the addition of Auxin to culture medium. Easy system construction by incorporation of one vector. Principle of Auxin-Inducible Degron (AID ) System This system expresses target protein fused to IAA17(hereafter called the aid tag) and F-box TIR1 protein derived from plant at the same time. The target protein is desighed in the cells so that protein fused to aid tag is expressed by only the cloning at MCS in the vector. We have two vectors which is fused to the N-terminal or C-terminal of target protein. This vector expresses at the same time both proteins of TIR1 and target protein fused to aid by using the internal ribosome binding sequence (IRES) derived from the encephalomyocarditis virus. AID plasmid protein In the presence of Auxin,the expressed TIR1acts in harmony with many intracellular factors as SCF ubiquitin ligase. In the presence of Auxin,TIR1 is activated and combines to aid tag fused to target protein. Then, aid tag fused to target protein is polyubiquitylated by ubiquitin and is resulted in rapid degradation by the proteasome.tir1 is inactive although Auxin is not present in the system. 1. Transfection of AID plasmid 2. Addition of Auxin

2 Rapid Degradation of Target Protein Control Control + Auxin + Auxin Degradation of GFP protein fused to aid after addition of Auxin. Cloned GFP fused with the aid plasmid was incorporated in 239 cells. Auxin of 500μM IAA was added to 293 cells and then cells were recovered to determine the expressed amount of GFP-aid protein by westernblotting. As you can see in the figure, degradation of the most of GFP-aid was found at 15mitutes after addition of IAA. Free Expression Control Expression control depending on the concentration of Auxin in 293 cells as described above was treated with different concentration of Auxin. You can find that GFP-aid fusion proteisn is expressed in the dose-dependent manner of Auxin. + Auxin Reversibility of Switch On/Off of Expression After degradation of GFP-aid in 293 cells, cells were transferred to the culture medium not containing Auxin and they were recovered to determine the expression. You can find that expression of GFP-aid is recovered after about 1 hour of withdraw. Control Time after removal of Auxin Example of Conditional Knock-Out of an Endogeneous Gene Tet-Off System (+ Tet) AID System (+ Auxin) Control system depending on tetracyclineresponsive TRE promoter has been used as a expression control system of CENP-H mrna. Therefore, degradation of protein depends on half time of various proteins in cells. On the other hand, AID system made it possible to control a rapid expression inhibition of target gene throughout a direct proteolysis of expressed protein. Protein depletion of CENP-H by AID system was compared with mrna depletion of CENP-H by the tetracyclinrepressive TRE promoter system in DT40 cells. Tetracycline treated or Auxin treated cells were collected at each time after the treatment Protein depletion of CENP-H in both cells was examined by immunofluoresence analysis (Green dots show protein of CENP-H and blue color shows nuclear DNA). CHNP-aid protein in DT40 treated with AID system disappeared within 30 minutes. On the other hand, CENP-H protein was still detected up to 24 hours when CENP-H mrna was depleted in DT40 cells. BR110517

3 PACKAGE CONTENTS: Set contents Quantity Concentration Storage Condition paid1.1-n Vector 20 ug 500ng/nL -20 C. Avoid freeze/thaw cycles. paid1.1-c Vector 20 ug 500ng/nL -20 C. Avoid freeze/thaw cycles. Indole-3-acetic acid (IAA) 1 ml 0.5 M -20 C. Avoid light. Anti-AID Tag Rabbit Polyclonal 100 ul C. Avoid freeze/thaw cycles. SHELF LIFE: One year from the date of receipt under proper storage conditions. DESCRIPTION: A protein of interest fused with a 25kD-degron expressed in animal cells from paid1.1-n Vector or paid1.1-c Vector can be conditionally controlled by addition of auxin such as IAA, which induces rapid degradation of the target protein. Together with the target protein, the plasmids express a plant specific F-box TIR1 protein, which makes an SCF TIR1 complex and works as an auxin-dependent E3 ubiquitin ligase for degradation of the target protein. The plasmids can be used for transient expression as well as for establishing stable cell lines in which the protein expression of interest can be controlled by auxin. PLASMID SIZE: paid1.1-n: 7438bp, paid1.1-c: 7450bp. ANTIBIOTIC RESISTANCE: Kanamycin (30-50 ug/ml for E. coli), G418 (800 ug/ml for HEK293 cells) REFERENCE: Nishimura K., Fukagawa T., Takisawa H., Kakimoto T. and Kanemaki M. An auxin-based degron system for rapid depletion of proteins in nonplant cells Nature Methods 2009, Vol.6, No.12, paid1.1-n Vector Information:

4 Multi Cloning Site (MCS) of paid1.1-n Restriction sites shown in MCS (EcoRV-EcoRI-PvuI-MluI-ScaI) are unique and suitable for cloning of the gene of interest. Description: paid1.1-n Vector encodes both the F-box TIR1 protein and a 25-kD degron (AID degron). When cloned in frame, your protein of interest would be expressed as a fusion protein with the degron at amino (N) terminus. Both TIR1 containing 9 repeats of Myc tag (TIR1-9Myc) and the fusion genes are driven from the immediate early promoter of cytomegalovirus (CMV prom) as a bicistronic mrna, the 3 terminus of which is processed by the poly-adenylation signal from the SV40 T antigen. The internal ribosome binding sequence (IRES) permits translation of the aid fused protein. The plasmid also contains an SV40 origin (SV40 ori) for replication in cells expressing the SV40 T antigen. For selection of E. coli and mammalian cells, the vector contains a kanamycin/neomycin-resistance cassette which confers resistance to kanamycin and G418, respectively. paid1.1-n Vector also contains both puc origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA replication. Location of Features: Human cytomegalovirus (CMV) immediate early promoter: TIR1-9Myc gene: IRES sequence: AID degron: Linker: MCS: SV40 early mrna polyadenylation signal: & ; mrna 3 ends: 4294 & 4306 f1 single-stranded DNA origin: Bacterial promoter for expression of Kan r gene -35 region: ; -10 region: Transcription start site: 4905

5 SV40 origin of replication: SV40 early promoter/enhancer: Kanamycin/neomycin resistance gene: Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal: puc plasmid replication origin: Propagation in E. coli: Transform E. coli (DH5α recommended) and select them on a LB plate containing ug/ml Kanamycin. For preparation of plasmid, grow E. coli in liquid LB containing ug/ml. paid1.1-c Vector Information: Multi Cloning Site (MCS) of paid1.1-c Restriction sites shown in MCS (EcoRV-EcoRI-PvuI-MluI-ScaI) are unique and suitable for cloning of the gene of interest. For efficient expression of the fusin protein of interest, the gene should be in flame with the ATG codon shown above.

6 Description: paid1.1-c Vector encodes both the F-box TIR1 protein and a 25-kD degron (AID degron). When cloned in frame, your protein of interest would be expressed as a fusion protein with the degron at carboxy (C) terminus. Both TIR1 containing 9 repeats of Myc tag (TIR1-9Myc) and the fusion genes are driven from the immediate early promoter of cytomegalovirus (CMV prom) as a bicistronic mrna, the 3 terminus of which is processed by the poly-adenylation signal from the SV40 T antigen. The internal ribosome binding sequence (IRES) permits translation of the aid fused protein. The plasmid also contains an SV40 origin (SV40 ori) for replication in cells expressing the SV40 T antigen. For selection of E. coli and mammalian cells, the vector contains a kanamycin/neomycin-resistance cassette which confers resistance to kanamycin and G418, respectively. paid1.1-c Vector also contains both puc origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA replication. Location of Features: Human cytomegalovirus (CMV) immediate early promoter: TIR1-9Myc gene: IRES sequence: MCS: Linker: AID degron: SV40 early mrna polyadenylation signal: & ; mrna 3 ends: 4306 & 4318 f1 single-stranded DNA origin: Bacterial promoter for expression of Kan r gene -35 region: ; -10 region: Transcription start site: 4917 SV40 origin of replication: SV40 early promoter/enhancer: Kanamycin/neomycin resistance gene: Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal: puc plasmid replication origin:

7 Propagation in E. coli: Transform E. coli (DH5α recommended) and select them on a LB plate containing ug/ml Kanamycin. For preparation of plasmid, grow E. coli in liquid LB containing ug/ml. Anti-AID Tag ( IAA17) Protein Antibody BACKGROUND: Detection of intracelluar AID plasmid (paid1.1-n Vector, paid1.1-c Vector or GFP AID NLC plasmid) expression is very critical for scientists who integrate them into the cells. Anti-AID tag (IAA17) protein antibody provides an excellent means for monitoring gene expression and protein localization in living cells. Polyclonal anti-aid tag (IAA17) antibody can detect AID tag (IAA17) on Western Blotting, Immunocytochemistry and Immunohistochemistry. SOURCE: The rabbit was immunized with recombinant AID tag (IAA17) protein. This antibody was purified from rabbit serum using affinity chromatography conjugated with purified AID tag (IAA17) protein. FORMULATION: This antibody solution contains PBS, 50% glycerol and 0.1% sodium azide as preservative. *Azide may react with copper or lead in plumbing system to form explosive metal azides. Please always flush plenty of water when disposing materials containing azide to drain. STORAGE: This antibody is stable for one year from the date of purchase when stored at -20. REACTIVITY: This antibody reacts with IAA17 on Western blotting, Immunocytochemistry and Immunohistochemistry. This antibody also detects AID tag conjugated with protein expressed in mammalian cells and Yeast cells on Western blotting, Immunocytochemistry and Immunohistochemistry.

8 APPLICATIONS: Western blotting: 1: 500~1:2,000* *Sensitivity of the immunodetection depends on reagents (secondary antibody, substrate, etc.) INTENDED USE: For Research Use Only. Not for diagnostic use. Western blot analysis of aid tag expression in mammalian cells (HEK293). Immunoblot detection of CENP-H-aid expression and knockdown in chicken DT40 cells. REFERENCE: Nature Methods Vol. 6, No.12, DECEMBER 2009,

9 Related products: Cat# Product Name Quantity APC001A paid1.1-n Vector 20 ug APC002A paid1.1-c Vector 20 ug APC003A IAAα 1 ml APC004A Anti-AID Tag Rabbit Polyclonal 100 ul APC005A paid1.1-n Vector and IAAα 1 set APC006A paid1.1-c Vector and IAAα 1 set APC007A paid1.1-n Vector and Anti-AID Tag Rabbit Polyclonal 1 set APC008A paid1.1-c Vector and Anti-AID Tag Rabbit Polyclonal 1 set APC009A paid1.1-n Vector, IAAα and Anti-AID Tag Rabbit Polyclonal 1 set A PC010A paid1.1-c Vector, IAAα and Anti-AID Tag Rabbit Polyclonal 1 set Notice to Purchaser: Use of Auxin-Inducible Degron (AID) System is covered by under Japan patent, which has been licensed to BioROIS Co., Ltd. Academic research institutions are granted an automatic license with the purchase of this product to use the AID system for internal, academic research purpose, which license specifically excludes the right to sell on otherwise transfer, the AID system on its component parts to third parties. No modification from to the protein coding sequence may be made without express written permission from BioROIS Co., Ltd. In accepting this license, all users acknowledge that the AID system is experimental in nature. BioROIS Co., Ltd. makes no warranties, express on implied of any kind, and hereby disclaims any warranties, representations on guarantees of any kind as to the AID system, patents on products. Manufacturer Distributor BioROIS Co., Ltd. TOYO 2CHOME, KOTO-KU, TOKYO, , JAPAN export@cosmobio.co.jp Phon e : FAX :

10 FQA about AID System Q1. Can any proteins be depleted by using the AID system? A1. Any proteins in cytoplasm and nucleus can be depleted by AID system. In the case of membrane proteins, however, aid-tag is required to be exposed inside of cytoplasm or nucleus because the aid-tag is the target of poly-ubiquitylation by the auxin-dependent manner. Proteins existing inside of mitochondoria can not be depleted by the AID system because there are no ubiquitin ligases in mitochondoria. nucleus aid Tag aid Tag Poly-ubiquitylation Rapid degradation by proteasomes Q2. Why can the AID system degrade target proteins so rapidly? A2. Because cells introduced the AID system sustains high ubiquitin ligase activity by constitutive expression of TIR1, aid-tag targeted ubiqutylation occurs as soon as aid-tagged proteins are expressed. Q3. Does not such high activity of ubiquitin ligase influence other endogeneous proteins? A3. Because ubiquitin ligase activity via TIR1 and anxin is highly specific to the aid-tag sequence, we expected that the TIR1/anxin dependent ubiquitin ligase activity had no influence to endogeneous proteins. As a matter of fact, our observation indicated no significant changes in expression level of other proteins. Q4. Which should we choose N-aid vector or C-aid vector? A4. We provide two vectors in order to fuse the aid-tag to N-terminus (N-aid) and C-terminus (C-aid) of a target protein of interest. You can choose a proper vector in

11 case you observe some influences to characters of the tagged protein. Q5. Is there something different between auxin (IAAα) provided by BioROIS and one available from other providers? A5. We provide auxin with stabilized status. But it requires storage at -20 C. Q6. What does AID system differ from widely used Tet On/OFF systems? A6. Because Tet ON/OFF systems control transcription level, depletion time of the target protein depends on stability of remaining mrna. However, AID system degrades the tagged-target protein itself very rapidly. Q7. Can we use in vivo systems introduced AID system? A7. Although many scientists request us in vivo systems, efforts to establish them are being undertaken. Q8. What research fields can the AID system be applied to? A8. We consider this system can be applicable to relations between gene functions and cell proliferation and development. In particular, it would be very useful for research fields such as oncology, aging, central nerve systems, infectious diseases, development of cells including ES and ips cells and so on. Manufacturer BioROIS Co., Ltd. Distributor TOYO 2CHOME, KOTO-KU, TOKYO, , JAPAN export@cosmobio.co.jp Phone : FAX :