NEMO [GST-tagged]

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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 ubiquitin chain binding domain activity of GST-NEMO was  validated through its ability to capture poly-ubiquitylated IRAK1  from a lysate preparation derived from IL-1 stimulated HEK293  cells. GST-NEMO was pre-incubated with Glutathione Sepharose  4B for 20 minutes at 4 °C followed by 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 SDS-PAGE/ Western  blotting (Lane 1). This sample was compared alongside similarlyderived  pull-downs from control reactions containing GST-NEMO wild-type versus  mutant (D311N) incubated in thepresence of lysates derived from either  IL-1 stimulated or nonstimulated HEK293 cells (Lanes 2-4).  Ubiquitylated IRAK1 was identified by Western Blotting using an  anti-IRAK1 antibody andsuch 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).