K-105

Material Data Sheet

Parkin Auto-Ubiquitination Kit

Price: $350.00
Catalog #: K-105

Parkin Auto-Ubiquitination Kit

Price: 
$350
Quantity: 
1 kit
Data Sheet: 
application/pdf iconK105_Parkin Auto-UB Kit_.pdf
Supplied: 
 ConcentrationVolume
10X E1 EnzymeX mg/ml (X μM)20 μl
10X UbcH7X mg/ml (X μM)20 μl
10X His6-ParkinX mg/ml (X μM)20 μl
10X Biotin-UbiquitinX mM20 μl
10X Reaction BufferX mM20 μl
Background: 
Mutations in the Parkin (PRKN2) gene are considered to be a major cause of autosomal recessive juvenile parkinsonism (AR-PJ). Parkin functions as an E3 ligase having an N-terminal ubiquitin-like motif and a C-terminal RING domain composed of two RING finger motifs separated by two IBR domains. Parkin can auto-ubiquitination itself and ubiquitinate various substrates (eg. CDCrel-1 and rel-2a, cyclin E, synphilin-1, the O-glycosylated form of α-synuclein (αSp22), PAel-R, FBP1, α/β tubulin, RanBP2, Hsp70, synaptotagmin XI) in an E2-dependent manner, targeting them for degradation. Parkin functions in conjunction E2 enzymes UbcH7 (E2-640), UbcH8 (E2-644) and UbcH13/Uev1 (E2-664). Parkin disease-associated mutations often affect E3 ligase activity through decreased E2 and/or substrate interactions which may thus result in the dysfunction of proteasomal degradation pathways and the neurotoxic accumulation of misfolded proteins.

This kit is designed as a control for to the conjugation of the ubiquitin to protein substrates in vitro, which requires the activities of the ubiquitin E1 (E-305), E2 UbcH7 (E2-640) and E3 Parkin (E3-150) activating enzyme. The E1 enzyme charges the ubiquitin by forming an ATP-dependent high energy thiolester bond. The activated ubiquitin is subsequently transferred to UbcH7 then to Parkin. The Parkin-S-Ub complex has the ability to both auto-ubiquitinate and transfer the ubiquitin to various substrates such as CDCrel-1 and rel-2a, cyclin E, synphilin-1, the O-glycosylated form of α-synuclein (αSp22), PAel-R, FBP1, α/β tubulin, RanBP2, Hsp70, synaptotagmin XI. Other E2s that have shown activity with Parkin are UbcH8 (E2-644) and UbcH13/Uve1a (E2-664). Alternatively labeled ubiquitins may be substituted for biotin-ubiquitin for visualization such as fluorescein-ubiquitin (U-590) and rhodamine-ubiquitin (U-600).

NOTE: Kit contains reagents sufficient for 10 x 20 μl reactions.
Concentration of components vary with Lot #.

Storage: 
Store at -80°C. Avoid multiple freeze/thaw cycles.

Literature

References: 

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