Invitrogen™ Ubiquitin Monoclonal Antibody (Ubi-1)

Description

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, a highly conserved protein that has a major role in targeting cellular proteins for degradation by the 26S proteosome, is synthesized as a precursor protein consisting of either polyubiquitin chains or a single ubiquitin fused to an unrelated protein. This gene encodes a fusion protein consisting of ubiquitin at the N terminus and ribosomal protein S27a at the C terminus. When expressed in yeast, the protein is post-translationally processed, generating free ubiquitin monomer and ribosomal protein S27a. Ribosomal protein S27a is a component of the 40S subunit of the ribosome and belongs to the S27AE family of ribosomal proteins. It contains C4-type zinc finger domains and is located in the cytoplasm. Pseudogenes derived from this gene are present in the genome. As with ribosomal protein S27a, ribosomal protein L40 is also synthesized as a fusion protein with ubiquitin; similarly, ribosomal protein S30 is synthesized as a fusion protein with the ubiquitin-like protein fubi. Multiple alternatively spliced transcript variants that encode the same proteins have been identified.