Ubiquitin Linkage Analysis and Ubiquitome Profiling

Ubiquitin is a small protein composed of 76 amino acids, featuring seven surface-exposed lysine (K) residues located at positions 6, 11, 27, 29, 33, 48, and 63. Subsequent ubiquitin moieties can be conjugated to any of these specific lysine residues on the preceding molecule. The specific linkage site dictates the unique three-dimensional conformation (topology) of the resulting polyubiquitin chain. Consequently, cellular recognition machinery interprets these distinct structural topologies to determine the downstream biological fate of the ubiquitinated target protein.

K48-linked chains drive proteasomal degradation and maintain cellular homeostasis, while K63-linked chains act in trafficking, signaling pathways, DNA repair and the regulation of protein-protein interactions. A degrader that raises the ubiquitination level of its target has therefore only demonstrated part of its mechanism, and the chain type establishes whether that modification corresponds to the degradation route the compound was designed to use. Amerigo Scientific supplies linkage-specific pull-down kits that identify the chain type on a protein of interest, chain-selective ELISA kits that quantify K48 and K63 ubiquitination in lysates, a complete panel of di-ubiquitin standards for characterizing deubiquitinase specificity, and a mass spectrometry kit for site-level and proteome-wide ubiquitome profiling.

Linkage-Specific UbiTest Kits

Chain linkage on a target protein has conventionally been determined by mass spectrometry or by Western blot with linkage-specific antibodies. Treating enriched material with a linkage-specific deubiquitinase before immunoblotting gives a more direct answer: an increased signal for the band corresponding to the unmodified protein of interest after treatment with a K48- or K63-specific deubiquitinase (DUB) shows that the protein carried chains of that linkage.

Tandem Ubiquitin Binding Entities (TUBEs)

Tandem Ubiquitin Binding Entities (TUBEs) are polyubiquitin capture reagents concatenating multiple ubiquitin-binding domains (UBDs), exploiting the avidity effect to achieve nanomolar-level affinity for polyubiquitinated proteins. This ultra-tight binding acts as a physical shield that protects the captured ubiquitin conjugates from endogenous deubiquitinating enzymes (DUBs) and proteasome-mediated degradation, ensuring their biochemical preservation without the need for chemical inhibitors. Furthermore, by engineering targeted combinations of UBDs, TUBEs can selectively recognize distinct polyubiquitin linkage topologies, enabling the precise isolation and characterization of chain-specific signaling complexes.

Product Range

We provide UbiTest kits that combine TUBEs-based enrichment with linkage-specific DUB digestion, in magnetic and agarose resin formats. Single-linkage kits supply either a K48-specific or a K63-specific DUB, while the profiling versions supply both, allowing the two linkages to be assessed side by side on the same enriched sample. All formats build on the pan-selective UbiTest workflow, whose TUBE1 resin binds all eight linkage types during the pull-down step.

Features

• Up to 1000-fold higher affinity for polyubiquitin compared to single UBA
• Avoids overexpression of epitope-tagged ubiquitin for pull downs
• Unambiguous determination of the ubiquitylation status of target proteins
• Can be multiplexed to examine several different targets in a single sample

In addition to the advantages shared by both formats, the magnetic format also has unique advantages, including: protecting polyubiquitinated proteins from degradation during cell lysis and subsequent pull down; facilitating quantitative determination of the ubiquitination percentage; and the magnetic beads enable the pull down efficient and minimize non-specific binding.

Chain-Selective Ubiquitin Linkage ELISA Kits

Compound series work needs linkage data in numerical form. Measuring K48 or K63 ubiquitination on target proteins directly in cellular and tissue lysates converts the question into a plate readout, which supports dose response, compound library processing and structure-activity relationship studies at a scale that immunoblotting cannot reach.

Amerigo Scientific provides chain-selective ELISA kits for relative and absolute quantitation of K48 and K63 ubiquitination on target proteins across treatments and conditions, as a quantitative and reproducible alternative to semi-quantitative Western blot analysis. Chemiluminescent and colorimetric versions are available, the latter reading at 650 nm and 480 nm on absorbance-based plate readers with day-to-day consistency that exceeds luminescence-based detection.

Features

• Replaces labor-intensive, semi-quantitative Western blot methods for examining K48 or K63 polyubiquitination and protein degradation.
• Offers quantitative, reproducible results.
• Facilitates high-throughput screening for compound library processing and structure-activity relationship (SAR) studies.

Ubiquitin Chain Standards

Deubiquitinases cleave the isopeptide bond between ubiquitin moieties, and their activity has traditionally been measured with ubiquitin carrying a fluorescent leaving group such as AMC or rhodamine at the C-terminus. Those substrates report cleavage of an amide bond, which stands as a proxy for isopeptidase activity, whereas di-ubiquitin molecules present the authentic isopeptide linkage and report linkage preference directly.

We provide a complete panel of all eight possible di-ubiquitin molecules, covering K6, K11, K27, K29, K33, K48, K63 and the C-to-N linear form, collected as a single kit for surveying the linkage specificity of individual deubiquitinases.

Ubiquitome Mass Spectrometry

Plate and blot assays report on a protein that has already been nominated. Mass spectrometry proteomics, particularly when combined with TUBE-based enrichment, opens the measurement to the ubiquitome as a whole, identifying ubiquitination sites on target proteins and extending to E3 ligase and substrate relationships that have yet to be described.

The mass spectrometry kit is designed for a simple, label-free and rapid ubiquitome workflow with reproducible results. In small molecule drug discovery, it reports quantitatively on how much target protein is degraded in the presence and absence of a candidate compound, which applies to PROTACs and molecular glues alike, and the same approach supports discovery of diagnostic biomarkers and clinical monitoring of disease progression.

Features

• Up to 1000-fold higher affinity for polyubiquitin compared to single UBA
• TUBEs have higher specificity and affinity than antibodies for IP applications
• Avoid overexpression of epitope-tagged ubiquitin for ubiquitin proteome enrichment
• TUBEs protect polyubiquitinated proteins from degradation and DUBs
• Specially designed polymeric magnetic beads offer efficient pulldown for MS analysis with lower non-specific binding
• Label free and extremely user-friendly
• Highly reproducible with higher proteome coverage compared traditional ubiquitin GG-remnant antibody approaches
• Enriches only ubiquitinated proteome compared to GG-remnant antibodies enrich peptides resulted from other post-translational modifications derived from trypsin digestion. (note: Trypsin digestion results in GG-remnant from ubiquitin like protein besides ubiquitin)