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NameCatalogue NumberSizePriceAdd to Basket
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Catalogue Number:66-1002-050Size:50 µgPrice:£250Add To Basket
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Specieshuman
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SourceE.coli
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Quantity50 µg
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Storage-70°C
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Concentration0.5 mg/ml
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Formulation50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
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Molecular Weight74.9kDa
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Stability12 months at -70°C; aliquot as required
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Protein SequenceAccession number: AAD38081. For full protein sequence information download the Certificate of Analysis pdf.
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QA; Protein IdentificationConfirmed by mass spectrometry.
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QA; ActivityUbiquitin 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).
Ubiquitin signals are decoded in cells by at least 200 ubiquitin binding proteins, which interact with different types of polyubiquitin chains and ubiquitin-like modifiers. These interactions induce conformational changes that allow these proteins to transmit the ubiquitin signal to effector proteins (Dikic et al., 2009). NEMO (NFκB Essential Modifier) is the protypic member of a family of proteins that interact with Lys63-linked and linear polyubiquitin chains (Nanda et al.,2011). NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. It is thought that this phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli (Kensche et al., 2012). NEMO is an integral component of the canonical IκB kinase (IKK) complex and is essential for the activation of IKKα and IKKβ, the protein kinase components of the complex. Mutations that abrogate binding of polyubiquitin chains to NEMO do not activate the IKK complex(Ea et al., 2006; Wu et al.,2006) and cause a severe immunodeficiency disease and greatly increased susceptibility to infection by bacteria of the tuberculosis family (Doffinger et al.,2001). NEMO also interacts with TANK, a component of the IKK-related kinases TBK1 and IKKε (Chariot et al., 2002).