Applications

Please Note: Optimal dilutions should be determined by each laboratory for each application.General Protocolsare available in the Technical Information section on our website.

Data Example

Western BlotView Larger

Detection of Human Ubiquitin by Western Blot.Western blot shows lysates of HCT-116 human colorectal carcinoma cell line. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human Ubiquitin Monoclonal Antibody (Catalog # A-104) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). Specific bands were detected for Ubiquitin at approximately 5-10 & 37-250 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

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Background: Ubiquitin

Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. Ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast. In mammals, four ubiquitin genes encode for two ubiquitin­ribosomal fusion proteins and two polyubiquitin proteins. Cleavage of the ubiquitin precursors by deubiquitinating enzymes gives rise to identical ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of ubiquitin to target proteins involves the formation of an isopeptide bond between the C­terminal glycine residue of ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi­step process that requires three enzymes: a ubiquitin­activating (E1) enzyme, a ubiquitin­conjugating (E2) enzyme, and a ubiquitin ligase (E3). Ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, ubiquitin was originally named ATP­dependent Proteolysis Factor 1 (APF­1). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post­endocytic sorting.

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