Ubiquitin is a highly conserved globular 76 amino acid protein of about 8.5 kDa molecular weight. It has a important role in the targeting of proteins for proteolytic degradation. Proteins to be degraded are covalently coupled to the C-terminus of ubiquitin by means of ubiquitin ligases. The ubiquitin itself is frequently also ubiquitinated, producing a polyubiquitin chain. The polyubquitinated complex is then recognized by a complex of degradative enzymes and structural proteins which together form the proteosome. Interestingly, ubiquitin also becomes covalently bonded to many types of pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so that ubiquitin antibodies are very useful for studies of these inclusions. For example the neurofibrillary tangles and paired helical filaments diagnostic of Alzheimer’s disease, the Lewy bodies seen in Parkinson’s disease, and Pick bodies found in Pick’s disease are all heavily ubiquitinated and can all be readily visualized with ubiquitin antibodies of appropriate specificity. This antibody, MCA-Ubi-1, was made in the University of Florida in 1987, and has been continuously on the market place since 1989, and is now being sold by several vendors. It has become widely used to study ubiquitinated inclusions seen in Alzheimer’s and other kinds of disease. MCA-Ubi-1 was raised against purified ubiquitin conjugated with glutaraldehyde to keyhole limpet hemocyanin. The clone was initially screened on ELISA of the immunogen, and subsequently tested on sections of Alzheimer brain. MCA-Ubi-1 was one of several clones which stained neurofibrillary tangles in frozen sectioned material strongly and specifically. Subsequent studies indicated that MCA-Ubi-1 is relatively insensitive to formalin fixation and so can be used on fixed histological sections of human brain for studies of Alzheimer’s and other neurodegenerative diseases. The antibody also works on paraffin embedded material. Consistent with the very high cross species sequence conservation of ubiquitin, MCA-Ubi-1 recognizes human, bovine, chicken, Drosophila, and C. elegans ubiquitin. MCA-Ubi-1 also works on western blots and can be used to study ubiquitinated proteins. It has also been successfully used in competitive ELISA. MCA-Ubi-1 is a mouse IgG1 with a κ light chain.
Blots probed with MCA-Ubi-1 of mono and K48 linked polyubiquitin (Boston Biotech, lane 1), monoubiquitin only (2), and 100μg total wet weight of homogenates of rat cerebellum, cortex and brain stem respectively (lanes 3-5). Material was run out on 20% SDS-PAGE and transferred electrophoretically to PVDF. MCA-Ubi-1 binds both mono and polyubiquitin and detects monoubiquitin in cell and tissue lysates.
Additional References;1. Fortun J, et al. Alterations in degradative pathways and protein aggregation in a neuropathy model based on PMP22 overexpression. Neurobiol Dis. 22:153-164 (2006).2. Boutajangout A, et al. Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer’s disease mutants of APP and presenilin-1. Neurobiol. Dis. 15:47-60 (2004).3. Wang DS, et al. Contribution of changes in ubiquitin and myelin basic protein to age-related cognitive decline. Neurosci. Res. 48:93-100 (2004).4. He CZ, Hays AP. Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis. J. Neurol. Sci. 217:47-54 (2004).5. Ungureanu D. et al. Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol Cell Biol. 22:3316-26 (2002)6. Wirbelauer C. et al. The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts. EMBO J. 19:5362-75 (2000).7. Harris KF. et al. Ubiquitin-mediated degradation of active Src tyrosine kinase. Proc Natl Acad Sci U S A. 96:13738-43 (1999).8. Sternsdorf T, et al. PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol Cell Biol. 19:5170-8 (1999).9. Marti A, Wirbelauer C, Scheffner M, Krek W. Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat Cell Biol. 1:14-9 (1999).CiteAb link to peer reviewed publications in which this antibody was purchased directly from us, here.Older peer reviewed publications making use of this antibody.Perry, G. et al. Proc. Natl. Acad. Sci. USA 84, 3033-3036 (1987)Shaw, G. and Chau, V. Proc. Natl. Acad. Sci. USA 85, 2854-2858 (1988)Hirano, S., et al. Cell 70: 293-301 (1992)Cuervo, A.M., et al. Mol. Biol. 9: 1995-2010 (1995)Sternsdorf, T., et al. J. Cell Biol. 139: 1621-1634 (1997)Tae-Wan Kim, et al. J. Biol. Chem. 272: 11006-11010 (1997)Verdier, F., et al. J. Cell Biol. 273: 28185-28190 (1998)Laroia, G., et al. Science 284: 499-502 (1999)Marti, A., et al. Nature Cell Biol. 1: 14-19 (1999)Sternsdorf, T., et al. Mol. Cell Biol. 19: 5170-5178 (1999)
1. Goldstein G, et al. Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.PNAS 72:11-5 (1975).2. Wilkinson K. The discovery of ubiquitin-dependent proteolysis. PNAS 102:15280-2 (2005).3. Perry G. et al. Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. PNAS 84:3033-6 (1987).4. Kuzuhara S, et al. Lewy bodies are ubiquitinated. A light and electron microscopic immunocytochemical study. Acta Neuropathol. 75:345-53 (1988).5. Murayama S. et al. Immunocytochemical and ultrastructural studies of Pick’s disease. Ann. Neurol. 27:394-405 (1990).6. Shaw G. Chau V. Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with differing distributions and solubility properties in Alzheimer brain. PNAS 85:2854-8 (1988).
This antibody has been widely used for about 30 years, though mostly sold through our OEM partners. Some of the papers which make use of the antibody supplied by EnCor can be found by searching Google Scholar for “MCA-Ubi AND antibody” or, if you are reading this online, go here.
for a very recent example of the use of MCA-Ubi-1 on western blots to visualize ubiquitin conjugates go to figure 4e in Gladcova C, et al. Mechanism of parkin activation by PINK1. Nature 559:410-414 (2018).
