K-240

Material Data Sheet

E6AP/E6 Ubiquitin Ligase Kit - p53 Substrate

Price: $495.00
Catalog #: K-240

E6AP/E6 Ubiquitin Ligase Kit - p53 Substrate

Price: 
$495
Quantity: 
1 Kit
Data Sheet: 

E6AP (E6-Associated Protein) (E3-230) is a HECT domain ubiquitin E3 ligase that ubiquitinates and mediates the proteasomal destruction of substrate proteins.  HECT domain ligases use an active site cysteine to accept charged ubiquitin from ubiquitin-E2 thioester complexes for subsequent transfer to substrate proteins; in this way HECT class ligases are distinct from most RING class ligases in that the latter facilitate transfer of ubiquitin from charged E2’s directly to substrate proteins without an E3-ubiquitin thioester intermediate.  E6 (Early protein 6) (AP-120) is a viral protein produced in cells infected with the Human Papillomavirus.  E6 forms a complex with the host cell E6AP generating a ligase activity that polyubiquitinates tumor suppressors p53 and p73 and targets them to the 26S proteasome for degradation.   As a result DNA damage and chromosomal instabilities increase, often leading to cell proliferation and cancer.  The E6/E6AP complex also targets other substrates for ubiquitination, such as TERT, BAK1, FADD, and pro-CASP8—none of which appear to be substrates for E6AP in the absence of E6.  This kit is designed for in vitro E6AP/E6-mediated ubiquitination of user-supplied substrates.  Ubiquitinated proteins can be used in downstream applications, or analyzed by Western blot using antibodies specific for the target protein.  A control substrate, His6-FLAG-p53 (SP-452) and detection antibody (MAB1355) are included in the kit.

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

Storage: 

Store protein components at -80oC. Avoid multiple freeze/thaw cycles. Loading Buffer may be stored at room temperature. Mg2+-ATP and α-p53 antibody may be stored at -20oC.

Literature

References: 

Beaudenon S. & Huibregtse J.M.  (2008)  BMC Biochem.  9 (Supp1): S4

Huang L., et al.  (1999)  Science   286: 1321-1326

Kim H.T. & Goldberg A.L. (2012)  Methods in Mol. Bio.  832: 653-660

Martinez-Noel G., et al.   (2012)   Mol. Cell. Biol.   32: 3095-3106

Mishra A., et al.  (2009)   J Biol Chem.  284: 10537-10545

Zanier K., et al. (2007)   Prot. Exp. & Purif.   51:  59-70

Zanier K., et al. (2010)   J. Mol. Biol.  396:  90-104