Invitrogen™ Ubiquitin Monoclonal Antibody (Ubi-1)

Mouse Monoclonal Antibody

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.