ITCH [GST-tagged]

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Species Reactivity: Human  

Source: E. coli expression  

Formula: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol  

Concentration: 0.5 mg/ml  

Molecular Weight: ~125.5 kDa  

Protein Sequence: Accession number: NP_1136713. For full protein sequence information download the Certificate of Analysis pdf.  

Quality Control Protein Identification: Confirmed by mass spectrometry.  

Quality Control Activity: E3 ligase assay:  The ubiquitin conjugating activity of GST-ITCH was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D3 (UbcH5c) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-ITCH for 60 minutes at 37 °C in the presence of ubiquitin, His-UBE1, His-UBE2D3 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-ITCH (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and GST-ITCH (with the exception of one species of approximately 140 MW observable in lane 3).