Ubiquitin Affinity Reagents （TUBEs）

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”）.

New Biotin-K63 TUBE 1 （UM304） displays a high sensitivity and specificity for polyubiquitin chains linked through K63. Biotin-K63 TUBE 1 is more sensitive and cost-effective for the detection of K63-linked polyubiquitylated proteins compared to traditional Western blot using chain-specific antibodies. Use Biotin K63-TUBE 1 for ligand blotting, affinity purification from lysates & biological fluids, and in situ detection.

Ubiquitin Derivatives and Chains

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. （www.progenra.com）.

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.

Isopeptidases – Assays

Ub/Ubl isopeptidases 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. Approximay 100 Ub/Ubl isopeptidases 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.

Isopeptidases – Enzymes

LifeSensors offers the largest collection of deubiquitinating （or deubiquitylating） enzyme （DUB） and Ubl isopeptidases. These enzymes are capable of recognizing and cleaving the isopeptide bond between ubiquitin （or Ubl） moieties or between ubiquitin （or Ubl） 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 isopeptidases can be utilized for HTS, determining Ub/Ubl specificity or Ub chain linkage type, as well as target substrate specificity.

Isopeptidases – Substrates

Deubiquitinating enzymes （DUBs） and UBL isopeptidases recognize and cleave the isopeptide bond between ubiquitin （or UBL） moieties or between ubiquitin （or UBL） and the conjugate protein. Measurement of the activity of these enzymes for kinetic study or inhibitor screening has been traditionally carried out using ubiquitin （or 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 isopeptidases: 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.

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 isopeptidases and conjugating enzymes. Along with classical inhibitors of Ub/Ubl isopeptidases 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.

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 ELISA

The UbiQuant™ ELISA is a sandwich assay in which total ubiquitin is captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent.  Unbound protein is 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.

‘UbiQuant ELISA is a great tool to determine the degree of specific protein ubiquitination and total ubiquitin levels in various cell fractions under different experimental conditions”.

Jayakumar Arumugam, Ph.D. University of Texas, M.D. Anderson Cancer Center

Ubiquitin Protein Microarray Services

Identification and characterization of the specificity of the substrate-enzyme interactions within the ubiquitylation and deubiquitylation pathways is a current challenge facing the field as a whole and for the individual researcher focused on an individual substrate or enzyme.

Combining unique protein microarray expertise and the most innovative catalog of ubiquitin research tools, LifeSensors’ Ubiquitin Protein Microarray Services provide a powerful microarray platform and straightforward approach to answering many questions at the forefront of ubiquitin research. Services include:

• E3 ID™ – Determine E3 ligases that ubiquitylate your substrate
• E3 Substrate ID™ – Identify substrates for your E3 ligase
• DUB ID™ – Determine DUBs that deubiquitylate your substrate
• DUB Substrate ID™ – Identify substrates for your DUB
• Custom Array Services  – Custom services can be developed for any question the arrays can answer

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.

*SilenciX® is a registered trademark of tebu-bio; technology licensed from the CEA.

Custom Ubiquitinated Peptides

Synthesis of custom monoubiquitinated peptides designed from your protein of interest

Choose the protein. . .
Choose the ubiquitination site. . .
We’ll deliver a monoubiquitinated peptide!

LifeSensors can provide custom monubiquitinated peptides from the sequence of your choosing. lsopeptide-linked ubiquitinated peptide reagents are produced using peptide synthesis technologies. Stepwise solid-phase synthesis and fragment condensation / chemical ligation approaches are employed in the synthesis of the ubiquitinated peptides. By utilizing these complementary methodologies for peptide synthesis, a large variety of native and labelled ‘isopeptide-linked’ ubiquitinated peptides can be generated for a variety of applications including:

•    DUB substrates for screening
•    Antigens for generation of site-specific ubiquitin antibodies
•    Structural studies of ubiquitin-binding domain recognition

In contrast to traditional DUB substrates, like ubiquitin-AMC and ubiquitin-rhodamine, which measure cleavage of amide bonds, utilization of a monoubiquitinated peptide as a DUB substrate measures cleavage of an isopeptide bond and allows determination of the influence of sequence proximal to the ubiquitinated lysine on specificity. Profiling activity of a panel of DUBs against a monoubiquitinated histone H2B peptide and a monoubiquitinated PTEN peptide reveals interesting differences in specificity:

Monoubiquitinated PTEN and histone H2B peptides are available from stock.

Length: peptides of almost any length can be synthesized
Quantity: µg to gram quantities
Purity: choose purity levels from 80-98%
Fluorescent modifications: examples include fluorescein, TAMRA, AlexaFluor®, or FRET pairs
Affinity tags: examples include biotin, His6, FLAG®, V5
Ubiquitin options: peptides can be synthesized with wild-type or mutant ubiquitins, including lysine to arginine substitutions or unnatural amino acids

Contact us at info@lifesensors.com or call 610-644-8845 for a quotation or more information.

Proteasome Research Tools

The proteasome is a 60 subunit, highly conserved protease complex, representing the products of ~35 genes. It is composed of a 20S core and one or two 19S regulatory particles （26S）. Most cellular proteins are marked for degradation by attachment of an ubiquitin chain, which leads to their rapid degradation by the 26S proteasome. The ubiquitin tag binds to the ‘lid’, a 19S particle, followed by disassembly of ubiquitin chains, protein unfolding and subsequent translocation into the 20S proteasome. The 19S proteasome contains 19 different proteins that have the ability to bind, deubiquitinate and unfold polyubiquitinated proteins. Unfolded proteins are further cleaved into short （3-15 amino acids） peptides.

SUMO Research Tools

E. Coli

Bacterial expression is the usual starting point for expression of heterologous proteins. Bacterial fermentations are inexpensive and can reach high cell densities resulting in high volumetric yields of the target protein. Our SUMO fusion technology is ideally suited for this system.

Mammalian

Mammalian cell expression has become the system of choice for production of complex, glycosylated biotherapeutic proteins such as antibodies, growth factors and fertility hormones. Our SUMOstar system has been designed for transient transfection studies in HEK293 or CHO cells.

Insect

Baculovirus/insect cell systems have found wide application for the expression of highly recalcitrant proteins such as protein tyrosine kinases. We have developed a SUMOstar-based system to support expression studies in this system.

Yeast

As with bacterial expression systems, yeast offer relatively inexpensive growth media and high density fermentation. Furthermore, yeast offer the capability of carrying out limited post-translational modifications such as disulfide bond formation and glycosylation. We offer two yeast expression systems, one for intracellular expression in S. cerevisiae and the second for extracellular expression in P. pastoris.