New Products - Quarter 4, 2005

SUMOylation Kit 
Product code: UW8955

This kit provides a means of generating SUMOylated proteins in vitro, by covalent linkage of the carboxy-terminal of SUMO-1, -2 or -3 to specific lysine residues on the target protein via isopeptide bonds, using the SUMOylation enzyme cascade. A control target protein is provided together with all other necessary components. SUMO specific antibodies are provided for detection of SUMOylated proteins via SDS-PAGE and western blotting.

Suggested uses / application:
1. SUMO-modification of specific proteins in vitro.
2. Demonstrating that novel proteins are potential targets for SUMOylation.  
3. Generation of substrates for deSUMOylating enzymes, such as SENP1 and SENP2 
4. Testing proteins for SUMO E3 ligase activity.  

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Ubiquitinylation (Ubiquitin Conjugation) Kit 
Product code: UW9920
 
This kit provides the means of generating a range of thioester-linked ubiquitin-conjugated E2 enzymes, utilizing the first two steps in the ubiquitin cascade, for use in the ubiquitinylation of E3 ligases and target substrate proteins. The reagents supplied also allow for the thioester formation and detection of E1-Ub and/or E2-Ub and the use of alternative (user supplied) E2 enzymes in E1 initiated/mediate reactions. Biotinylated ubiquitin is provided for sensitive detection with streptavidin-linked enzymes via SDS-PAGE and western blotting.

Suggested uses / application:
1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase. 
2. Activation of ubiquitin for thioester conjugation to novel E2 enzymes.
3. Use of cell lysate or crude fractions/preparations as source of E3 ligases. 
4. Substrate (target) independent in vitro ubiquitinylation reactions. 

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TPPII Complex (Tripeptidyl peptidase II), (human erythrocyte)
Product code: PW9660

In eukaryotes, tripeptidyl peptidase II is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway in the breakdown of most nuclear and cytoplasmic proteins.  TPPII is an amino peptidase belonging to the subtilase family, removing tripeptides from the free N-terminus of oligopeptides.  More specific roles for TPPII include its role in major histocompatibility complex (MHC) class I antigen processing where it appears to be an important intermediate between the proteasome and the rest of the peptidase pool involved in trimming of larger fragments to antigenic peptides and free amino acids.  Ongoing structural studies with material from a number of species show native TPPII to be a high molecular mass homomeric protein where the subunit (138 kDa) forms a large cylinder-like and well-organized complex (Mr>10⁶), approximately 17 nm in diameter and 50 nm in length.  Like the proteasome, the enzyme appears to have a ubiquitous distribution.  Substrate specificity is fairly broad and is highly dependent on a free N-terminus. TPPII may be able to substitute for some metabolic functions of the proteasome.  

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Di-ubiquitin (Ub₂), K⁴⁸-linked
Product code: UW9800

Di-ubiquitin may be used to aid investigation of ubiquitin binding entities with chains of defined length and character. Ubiquitin mutants [K⁴⁸C] and [D⁷⁷] were expressed in BL21 (λDE3) E. coli. Proteins were purified by cation exchange chromatography. Di-ubiquitin was synthesised by linking [K⁴⁸C]ubiquitin and [D⁷⁷]ubiquitin in the presence of ubiquitin conjugating enzyme E2-25K (UbcH1) and human ubiquitin activating enzyme E1. 

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RanGAP1 Fragment (241-360), GST-tagged (human, recombinant)
Product code: UW9755

RanGTPase-activating RanGAP1 was the first protein shown to be post-translationally modified with SUMO.  In higher eukaryotes, the cellular localization of RanGAP1 is regulated by SUMOylation of its C-terminal domain. The target lysines of RanGAP1, as well as the C-terminus of mature SUMO-1, lie within mobile regions of the two proteins. Upon SUMOylation, RanGAP1 and SUMO-1 behave as “beads-on-a-string” joined by a flexible isopeptide tether and their structures and local dynamic features do not change significantly beyond the site of this covalent linkage. RanGAP1 acts as a very good control substrate for use in SUMOylation assays producing a product with a single SUMO modification as shown above.

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SENP1 Fragment (415-643), GST-tagged (human, recombinant)
 Product code: UW9760

SENP1 fragment has been reported to co-localize with a herpes virus protein ICP0 during early infection.  This co-localization may be involved in the ICP0-induced loss of SUMO-1-modified PML in infected cells.  Characterization of the localization and regulation of SENP1 has also been reported.  SENP1 localization may also be influenced by the expression of proteins that are targets of SUMO-1 modification, such as HDAC4 and PML, which affect the relative levels and localization of SUMO-1 conjugates within the cell. This fragment has full de-SUMOylating activity.  

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SENP2 Fragment (368-549), GST-tagged (human, recombinant)
Product code: UW9765

SENP2 was discovered both through its homology to other SUMO proteases and its interactions with murine Axin, a regulator of the Wnt signalling pathway.  When overexpressed in tissue culture cells or under in vitro conditions, the murine SENP2 homologue (Smt3IP2) cleaves conjugates of SUMO-1, SUMO-2, and SUMO-3.  Full-length human SENP2 associates with nuclear pores in a manner similar to Ulp1 in yeast.  This association occurs exclusively with the nuclear face of the pore and requires sequences near the N-terminus of SENP2.  This fragment has full de-SUMOylating activity.

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NEDP1, His₆-tagged (human, recombinant)
Product code: UW9770

NEDP1, a human NEDD8-specific protease, is highly conserved throughout evolution and equivalent proteins are present in yeast, plants, insects, and mammals.  NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates. Inhibition studies and mutagenesis indicate that NEDP1 is a cysteine protease with sequence similarities to SUMO-specific proteases and the class of viral proteases typified by the adenovirus protease. In vivo NEDP1 deconjugates NEDD8 from a wide variety of substrates including the cullin component of SCF-like complexes.  Thus NEDP1 is likely to play an important role in ubiquitin-mediated proteolysis by controlling the activity of SCF complexes.  

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