K-270

Material Data Sheet

ITCH/AIP4 Ubiquitin Ligase Kit

Price: $395.00
Catalog #: K-270

ITCH/AIP4 Ubiquitin Ligase Kit

Price: 
$395
Quantity: 
kit
Data Sheet: 

The mammalian Itchy homolog, or ITCH, (also known as Atrophin-1-interacting protein 4 or AIP4) is a HECT domain class ubiquitin E3 ligase. ITCH ubiquitinylates the phosphorylated form of Dishevelled protein and promotes its degradation via the Ubiquitin Proteasome System, thereby inhibiting canonical Wnt signaling. The absence of ITCH has been shown to cause severe autoimmune disease in mice. Recent studies have identified multisystem autoimmune disease and morphologic and developmental abnormalities in human patients with ITCH deficiency, thus underscoring the importance of ITCH in many cellular processes. This ligase may catalyze K29-, K48-, and/or K63-linked polyubiquitin chain formation on a variety of reported targets.

This kit is designed for in vitro ITCH-mediated ubiquitinylation of user-supplied substrates. Ubiquitinylated proteins can be used in downstream applications, or analyzed by Western blot using antibodies specific for the target protein. A detection antibody is included in the kit to monitor autoubiquitinylation of ITCH.

Kit contains reagents sufficient for 10 x 30 μl reactions and 5 Western Blots (mini-gel format).

Product Information

Western Blot Data: 
Supplied: 
E1 enzyme, E2 enzyme (UBE2L3), ITCH, ubiquitin solution, Reaction Buffer, Mg2+-ATP, α-ITCH primary antibody and Loading Buffer

Use & Storage

Use: 

ITCH is robustly autoubiquitinylated using the reagents and protocol conditions supplied in this kit. Reactions are complete within 60 minutes at 37°C, resulting in ubiquitinylation of 75% or more of the ligase. The kit protocol is designed for reaction termination with SDS-PAGE Sample Buffer; therefore, proteins are denatured and typically not suitable for further enzymatic manipulation. If autoubiquitinylated ITCH or ubiquitinated user-provided substrate is to be utilized in further reactions prior to SDS-PAGE analysis, ubiquitination reactions may be terminated by the addition of EDTA (10 mM final) plus DTT or βME (5-10 mM final) if compatible with downstream experimental protocols. SDS-PAGE gels, PVDF membrane, blocking reagent, antibodies and protocols provided or referenced in this kit have been chosen following careful testing at Boston Biochem. Modifications to the protocol or selection of alternative reagents may require assay optimization by the end-user. Further information is available via techsupport@bostonbiochem.com.

Storage: 

Store protein components at -80ºC. Avoid multiple freeze/thaw cycles. Loading Buffer may be stored at room temperature. Mg2+-ATP and α-ITCH antibody may be stored at -20°C.

Literature

References: 

  1. Wei W., et al.  (2012)  Mol. Cell Biol.  32: 3903-12
  2. Lohr N.J., et al.  (2010)  Am. J. Hum. Genet.   86: 447-453
  3. Yang C., et al.  (2006)  Mol. Cell     21: 135-141