Ubiquitin Research Tools

Anti-Ubiquitin Detection and Isolation Tools

LifeSensors has developed TUBE (Tandem Ubiquitin Binding Entity) technology for the detection, characterization and isolation of polyubiquitylated proteins from cells and tissue extracts.  TUBEs have 100 to 1000-fold higher affinity for polyubiquitin chains compared to monomer ubiquitin binding domains (UBDs). TUBEs both stabilize and bind to ubiquitylated proteins, serving as an indispensable tool for ubiquitologists.

Our versatile collection of TUBEs includes reagents for isolation/pulldown polyubiquitin conjugates (Agarose-TUBEs, GST- and His6-TUBEs). For immunohistochemistry, use Fluorescent TUBEs conjugated to either fluorescein or TAMRA. Use Biotin-TUBEs for the detection of polyubiquitylated proteins by ligand blotting (“Far Western”).

UbiTest: Awaken The Wolf

The most common method to investigate protein ubiquitylation is through immunoprecipitation and immunoblot analysis; however, the substrate antibody may interact differently with a ubiquitylated substrate due to epitope masking, reduced affinity, or changes in selectivity. A more definitive method for demonstrating protein ubiquitylation is to couple immunoprecipitation with digestion by a broad spectrum deubiquitylase prior to immunoblot analysis. An increased signal for the protein of interest (POI) after DUB treatment is a clear indication that the protein was ubiquitylated even if there was no clear reactivity in the untreated sample. To avoid potential problems arising from changes in immunoreactivity of the POI, it is best to pull-down the protein with an anti-ubiquitin reagent. We have built this kit around the use of Tandem Ubiquitin Binding Entities (TUBEs), which are engineered tandem UBAs with dissociation constants for tetra-ubiquitin in the nanomolar range. The nanomolar affinity of TUBEs for polyubiquitinated proteins allows high efficiency isolation and characterization of these proteins from cell lines and tissues. TUBE1 has been demonstrated to bind to all 7 linkage types.

Conjugation Assays

The conjugation of ubiquitin to substrates is a multi-step, multi-enzyme process. LifeSensors is committed to supplying high throughput, facile, and robust assays for measuring the activity of Ub conjugating enzymes. These assays work with a wide range of E3 ligases including members of the three major classes of E3 ligases (simple RING class, SCF RING class, and HECT class).  Our E3LITE Customizable Ubiquitin Ligase Kit, as well as the E2 profiling kit based on it, was developed in collaboration with and licensed from Progenra, Inc.

Conjugating Enzymes

Ubiquitin/Ubl conjugating enzymes are responsible for the selective modification of substrates. LifeSensors supplies a wide assortment of these enzymes to aid researchers in their efforts to understand the specificity and selectivity of this complex enzyme pathway.

Deubiquitylases – Assays

Ub/Ubl deubiquitylases play key regulatory roles in a variety of disease states including cancer, diabetes, and infectious disease. As such, they represent an attractive class of novel targets for therapeutic development. Approximately 100 Ub/Ubl deubiquitylase have been identified in the human genome, however relatively few have been fully characterized. LifeSensors’ offers a wide range of HTS amenable assays substrates for characterizing these proteases and discovering modulators of their activity.

Deubiquitylases – Enzymes

LifeSensors offers the largest collection of deubiquitinating (or deubiquitylating) enzyme (DUB) and ubiquitin like moieties (Ubl) isopeptidases. These enzymes are capable of recognizing and cleaving the isopeptide bond between ubiquitin moieties or between Ubl moieties and the target protein. Five classes of DUBs exist based on sequence homology. These families include the ubiquitin C-terminal hydrolases (UCH), the ubiquitin-specific processing proteases (USPs), the Machado-Joseph Disease domain proteases, the Otubain proteases (Otu), and JAMM domain proteases. LifeSensors DUBs and Ubl deubiquitylase can be utilized for HTS, determining Ub/Ubl specificity or Ub chain linkage type, as well as target substrate specificity.

Deubiquitylase – Substrates

Deubiquitylating enzymes (DUBs) and ubiquitin UBL  isopeptidase recognize and cleave the isopeptide bond between UBL moieties or between UBL and the conjugate protein. Measurement of the activity of these enzymes for kinetic study or inhibitor screening has been traditionally carried out using UBL conjugated to fluorescent leaving groups (AMC or rhodamine) at the C-terminus. While these are suitable substrates for many deubiquitinating enzymes, the cleavage of the amide bond serves as a proxy for the true activity of deubiquitylase: cleavage of an isopeptide bond. LifeSensors has recently produced internally-quenched fluorescent diubiquitins, which consist of two ubiquitins singly labeled with a fluorophore and a fluorescent quencher. The cleavage and separation of the isopeptide bond between the labeled ubiquitins results in a fluorescent signal. The IQF-diubiquitins therefore provide sensitive fluorescent substrates for continuous measurement of the isopeptidase activity of DUBs. Lifesensors currently offers IQF-diubiquitin substrates linked through K11, K48 and K63.

Proteasome Research Tools

SilenciX Ubiquitin Pathway Knockdown Cell Lines

The first generation of RNAi tools, siRNA and basic plasmids expressing shRNA, reveal certain limitations and off-target effects. Furthermore, successful RNAi experiments currently involve significant optimization requiring additional resources.

SilenciX® stable knockdown cell lines have been developed based on a unique siRNA delivery system developed by tebu-bio Laboratories* providing improved efficacy and stability of RNAi knockdown. Based on EBV-replication, this loss-of-function model ensures chromosome-like replication of the expression vector in a limited and constant number without integration in the genome. This eliminates off-target effects and saturation of the RNAi machinery resulting in specific and homogeneous knockdown of the desired target.

LifeSensors is offering SilenciX®, gene-specific ready-to-use knock-down cell lines ‘silencing’ deubiquitylases (DUBs) and other proteins of interest, including DNA repair-related genes, providing invaluable tools for loss-of-function approaches.

Ubiquitin Derivatives and Chains

Ubiquitin ELISA

UbiQuant™ is a sandwich-type ELISA assay in which total ubiquitin is captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent.  Unbound proteins are removed by washing and then an anti-ubiquitin monoclonal antibody (VU-2) is added.  After removing unbound VU-2, bound antibody is measured using an enzyme linked anti-mouse antibody.

Ubiquitin/Ubl Pathway Antibodies

Antibodies can be used in a wide range of applications including Western blotting, ELISA assays, immunofluorescence and immunoprecipitation. LifeSensors offers quality antibodies recognizing many of the enzymes involved in protein ubiquitylation to aid researchers in their quest to uncover novel protein functions.

Ubiquitin/Ubl Pathway Inhibitors

Increasingly, associations are being made between the ubiquitin system and many human diseases including cancer, neurodegeneration, metabolic disorders and viral infections. Proteolysis mediated by the ubiquitin-proteasome system is regulated by different enzymes, which recently became an attractive target for drug discoveries. LifeSensors offers a number of inhibitors enabling researchers to decipher the function of Ub/Ubl deubiquitinase and conjugating enzymes. Along with classical inhibitors of Ub/Ubl deubiquitinase such as Ub-vinyl methyl ester and –aldehyde, LifeSensors has a number of small molecule inhibitors. Our new addition includes inhibitors of Ub/Ubl conjugating enzymes. With an evergrowing interest in proteasomal activity that has been linked to cancer, inflammatory and autoimmune diseases, we now offer a collection of proteasome inhibitors. Several inhibitors including bortezomib are already used in the treatment of multiple myeloma. The most commonly used laboratory inhibitors, lactacystin and MG132 are now available in different quantities.

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