UM413: K63 Linkage Specific UbiTest – Agarose TUBE Elution Kit

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. Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through mass spectrometry (MS) or by Western blot using linkage specific antibodies. LifeSensors has developed a more definitive method for demonstrating the poly-ubiquitination linkage of a protein by using a linkage-specific DUB prior to the immunoblot analysis. An increased signal for the  band corresponding to the unmodified POI after a K63-specific DUB treatment is a clear indication that the protein was K63 poly-ubiquitinated.

Kit Components:

Tube 1	TUBE 1 (High Capacity Magnetic Beads)– 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	Neutralization buffer (10X) – 0.5 mL
Tube 5	Broad Spectrum DUB – 25 µg, 10 µM
Tube 6	A K63-linkage specific DUB – 35 µg, 10 µM

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