Cellular Ubiquitination Quantification and Confirmation

Once a compound has been shown to drive ubiquitination in a reconstituted reaction, the question moves into cells: is the target protein polyubiquitinated in the lysate, and how does that level change with compound and dose. Immunoprecipitation followed by Western blot is the conventional route to this answer, and it is limited by how the substrate antibody behaves toward the modified forms of the substrate, since epitope masking, reduced affinity and changes in selectivity all affect the immunoblotting step.

Amerigo Scientific provides plate-based sandwich assays that quantify the polyubiquitinated forms of a protein of interest directly in cell lysate, a matching assay for absolute measurement of total ubiquitin, and tandem ubiquitin binding entities (TUBEs)-based pull-down kits that establish whether a given protein carries polyubiquitin at all.

Ubiquitination Confirmation by TUBE Pull-Down

Coupling a pull-down with digestion by a broad spectrum deubiquitinase gives a definitive answer on whether a protein is ubiquitinated. Polyubiquitinated proteins are captured, the captured material is treated with the deubiquitinase (DUB), and an increased signal for the protein of interest after that treatment shows that the protein was ubiquitinated, even where the untreated sample gave weak reactivity. Capture is performed with Tandem Ubiquitin Binding Entities, engineered tandem ubiquitin-binding domains whose dissociation constants for tetra-ubiquitin sit in the nanomolar range, so the enrichment step is independent of the substrate antibody.

We provide UbiTest kits in magnetic and agarose resin formats, each supplied with TUBE1 resin, wash, elution and neutralization buffers, and a broad spectrum DUB. TUBE1 has been shown to bind all eight polyubiquitin linkage types, which keeps the pull-down open to whichever chain type a degrader produces.

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

Target Protein Polyubiquitination Quantification

Ranking degraders on potency requires a number rather than a band. Capturing the polyubiquitinated fraction of a lysate on a plate and then detecting the protein of interest within that fraction converts the measurement into a quantitative readout, which makes dose response and compound comparison practical at plate scale.

The UbiQuant S assay is designed for measuring the polyubiquitination level of a protein of interest in cell lysate, as a quick and quantitative alternative to immunoprecipitation followed by Western blot. 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. Coated plates are also supplied on their own for laboratories that provide their own antibodies and detection reagents.

Low-Abundance Target Quantification

Degrader programs frequently work on targets that are present at low copy number, where the polyubiquitinated fraction represents a small share of an already small pool. Detection sensitivity, rather than assay logic, sets the limit in these experiments.

Amerigo Scientific provides the UbiQuant Ultra plate for assessing polyubiquitination levels of low abundance proteins of interest in cell lysate, on the same capture and detection principle as UbiQuant S, in chemiluminescent and colorimetric versions. UbiQuant™ Ultra Plate only includes the coated plate.

Features

• Polyubiquitination measurement for low abundance targets
• Detection of small changes in ubiquitination state
• Extension of UbiQuant S workflows to weakly expressed proteins

Total Ubiquitin Quantification

The polyubiquitination level of a single target is easier to interpret alongside the size of the ubiquitin pool it draws on, since compound treatment, proteasome inhibition and cellular stress all shift the total amount of ubiquitin in a lysate. We provide UbiQuant™, a sandwich assay for absolute measurement of ubiquitin in cell lysate. Total ubiquitin is captured with a proprietary ubiquitin binding reagent, an enzyme-linked anti-ubiquitin monoclonal antibody generates the signal, and a ubiquitin standard curve converts that signal into a direct measurement of concentration. Concentration results are obtained in µg/mL and not in arbitrary units.

Features

• Absolute quantification against a ubiquitin standard curve
• Sandwich format with proprietary capture reagent
• Enzyme-linked anti-ubiquitin monoclonal detection
• Plate reader readout