13-1600 recognizes ubiquitin, both conjugated and unconjugated. It reacts with a single chain 8.5 kDa protein. The ubiquitin molecule appears to be present in all eukaryotic cells and has an identical primary structure in all animals. Ubiquitin is present in the nucleus, cytoplasm, and on cell surface membranes.
This antibody is suitable for immunohistochemical staining of alcohol- or paraformaldehyde-fixed, paraffin-embedded or frozen tissue sections. Heat-induced epitope retrieval with citrate buffer, pH 6.0, is required for specific staining of formalin-fixed, paraffin-embedded tissue sections. To stain, incubate 30-60 minutes at room temperature or overnight at 4°C. This antibody may also be used in ELISA and has been successfully used in western blot analysis of ubiquitinated proteins in mouse thymocytes.
To perform western blotting, prepare lysis buffer in 10 mM N-Ethylmaleimide to inhibit ubiquitin-conjugating enzymes. N-Ethylmaleimide inactivates certain enzymes by blocking free sulfhydryls. After electrophoresis and transfer, pre-incubate transferred membranes in denaturing buffer (6 M guanidine-HCl, 20 mM Tris-HCl, pH 7.5, 5 mM betamercaptoethanol, 1 mM PMSF) for 30-60 minutes at 4°C, followed by extensive PBS washing.
Ubiquitin is a multifunctional and extremely highly conserved 76-amino acid polypeptide of ~8.5 kDa. An unusual property of ubiquitin is that it can become linked to a lysine e-amino group in another protein to produce a covalent ubiquitin-conjugate, and can affect proteasomal degradation of the protein it is bound to, or mediate interactions with other proteins related to post-translational modifications. Such conjugates are usually very unstable, as they are recognized by a specific ubiquitin-dependent protease, and rapidly degraded. Ubiquitin therefore appears to target such proteins for rapid degradation. The degradation of cellular regulatory proteins by the ubiquitin pathway is important as it controls cellular growth and proliferation. Ubiquitin-dependent proteolysis occurs after a covalent attachment of the peptide to a lysine residue of a protein, which involves three enzymatic reactions: E1, E2, and E3. The first reaction involves ubiquitin-activating enzyme. The third reaction uses enzyme ubiquitin ligase (E3) to transfer the activated ubiquitin from E2 to a lysine residue on a protein, or directly transfers the ubiquitin from E2 to the substrate. There are also examples of stable ubiquitin conjugates, including histone H2a and some unusual forms of actin. In the last few years several groups have noted that a variety of pathological inclusions found in many human disease states exist as stable ubiquitin conjugates. These inclusions include the neurofibrillary tangles of Alzheimer's disease and progressive supranuclear palsy, Pick bodies of Pick's disease, Lewy bodies of Parkinson's disease, Mallory bodies of alcoholic liver disease, Rosenthal fibers of Alexander's disease, and the inclusion bodies in inclusion myositis and oculopharyngeal muscular dystrophy. Such inclusions can be visualized efficiently with antibodies to ubiquitin. Why ubiquitin is found in this variety of inclusions is not currently known, although it seems likely that the cell has tried and failed to remove these insoluble inclusions via the ubiquitin-dependent proteolysis pathway.
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Protein Aliases: 40S ribosomal protein S27a; Chap1; DSK2 homolog; epididymis luminal protein 112; hPLIC-2; HRIHFB2157; PLIC-2; Protein linking IAP with cytoskeleton-2; Ubiquilin 2; ubiquitin and ribosomal protein S27a; ubiquitin C; ubiquitin carboxyl extension protein 80; ubiquitin-40S ribosomal protein S27a; ubiquitin-CEP80; Ubiquitin-like product Chap1/Dsk2; ubiquitin/ribosomal protein
Gene Aliases: 0610006J14Rik; BOS_11463; CEP80; HEL112; S27A; Uba52; UBA80; Ubb; UBC; UBCEP1; UBCEP80