The UbiTest kit determines whether or not a target protein is poly-ubiquitinated. This powerful platform is an improvement on the most common method for investigating protein ubiquitination: immunoprecipitation and Western blot analysis. However, the substrate antibody often interacts differently with the poly-ubiquitinated forms of the substrate in the immuno-blotting step. This is due to epitope masking, reduced affinity, or changes in selectivity. A more definitive method for demonstrating protein ubiquitination is to couple immunoprecipitation with digestion by a broad spectrum deubiquitinase (DUB) prior to immunoblot analysis. An increased signal for the protein of interest (POI) after DUB treatment is a clear indication that the protein was ubiquitinated even if there was no clear reactivity in the untreated sample. To avoid potential problems arising from changes in immunoreactivity of the POI, the UbiTest assay utilizes TUBEs to pull-down the poly-ubiquitinated proteins. TUBEs (Tandem Ubiquitin Binding Entities) are engineered tandem ubiquitin-binding domains with dissociation constants for tetra-ubiquitin in the nanomolar range. TUBE1 has been demonstrated to bind to all 8 linkage types.
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Tube 1 — Magnetic TUBE1 – 1 mL of slurry, 100 µL slurry is recommended for 1-2 mg of total cell lysate pull down.
Buffer: PBS, pH 7.2, 20% ethanol
Storage: Please keep the magnetic TUBE1 at 4°C. Do not centrifuge, dry or freeze the beads
Tube 2 — Wash buffer – 2 mL
Tube 3 — Elution buffer – 1 mL
Tube 4 — 10X Neutralization buffer (Add β-mercaptoethanol to a final concentration of 10 mM before use, e.g. add 5 µl of 1 M β-mercaptoethanol stock solution) – 0.5 mL
Tube 5 – Broad Spectrum DUB – 25 µg, 10 µM