NEMO (D311N) [GST-tagged]

Ubiquigent

Catalog No: UBI-66-1013-050

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Species Reactivity: human  
Source: E.coli  
Formula: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol  
Concentration: 0.5 mg/ml  
Molecular Weight: 74.9kDa  
Protein Sequence: Accession number: AAD38081. For full protein sequence information download the Certificate of Analysis pdf.  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control Activity: Ubiquitin Binding Domain Activity: The lack of GST-NEMO (D311N) ubiquitin chain binding domain activity was determined through its inability to capture polyubiquitylated IRAK1 from a lysate preparation derived from IL-1 stimulated HEK293 cells. GST-NEMO (D311N) was pre-incubated with Glutathione Sepharose 4B for 20 minutes at 4 °C followed by an incubation for 2 hours at 4 °C with 2mg IL-1 stimulated HEK293 cell lysate. The binding reaction was then centrifuged and the pellet analysed by SDSPAGE/Western Blotting (Lane 2). This sample was compared alongside similarly derived pull-downs from control reactions containing GST-NEMO wild-type versus mutant (D311N) incubated in the presence of lysates derived from either IL-1 stimulated or non-stimulated HEK293 cells (Lanes 1-4). Ubiquitylated IRAK1 was identified by Western Blotting using an anti-IRAK1 antibody and such species were observed only in the pellet sample derived from a binding reaction containing wild-type GST-NEMO and IL-1 stimulated HEK293 cell lysate (Lane 1).