HeLa cell cultures were stained with MCA-5A12 antibody (green). The antibody stains spindle poles and mitotic spindles at anaphase (top 2 panels) and concentrates on the midbody between the two daughter cells during telophase (bottom 2 panels). It is therefore a useful marker of dividing cells. Cells were counterstained with our chicken polyclonal antibody to Vimentin CPCA-Vim
in red. Blue is a DNA stain.
|Left: Western blot analysis of MCA-5A12 in HeLa cells. Blot of HeLa cells treated with 100ng/ml nocodazole for 18 hours was probed with MCA-5A12. Nocodazole is a microtubule polymerization inhibitor which induces cells to halt at the G2/M phase and also induces Aurora A expression. The MCA-5A12 monoclonal binds strongly to a band at about 46 kDa, which is Aurora A and also shows binding to a band at 38 kDa. Right: Blots of recombinant human Aurora A, B and C were probed with MCA-5A12. We conclude that this antibody reacts with the Aurora A and Aurora B kinases and that the 38 kDa band seen in the HeLa extract is Aurora B. This is also consistent with staining of both centrosomes and midbodies seen on HeLa cells.
||Anti-Aurora A/B kinase
||Mouse Monoclonal to Aurora A/B kinase.
||46 kDa, 38 kDa
||Full length recombinant human Aurora A protein expressed in and purified from E. coli.
||Antibody is supplied as an aliquot of 1 mg/mL of affinity purified antibody.
||Human, horse, cow, pig, chicken, rat, mouse
||Western blot, ICC/IF, IHC
|Suggestions for use
||Western blots: 1:1,000-1;2,000.
ICC/IF or IHC: 1:1,000-1:2,000.
Aurora proteins are a family of serine/threonine protein kinases that play a key role in the regulation of cell division. The first Aurora kinase was discovered in Drosophila
(1). Mutations of this kinase cause monopolar spindles surrounded by kinase, and the appearance of this was reminiscent of the Aurora borealis at the poles of the earth (1). Mammals contain three Aurora kinases named Aurora A, Aurora B, and Aurora C. All three contain a regulatory domain in the N terminus which is quite different between the three molecules followed by a catalytic serine/threonine kinase domain which is almost identical between the three. To download a sequence alignment of the 3 human Aurora proteins go here
. As a consequence antibodies raised against one Aurora family member frequently cross-react with other family members. There is a short C-terminal peptide which is also variable between the three molecules (2). Aurora A is required for centrosome duplication, entry into mitosis, formation of bipolar spindle and mitotic checkpoint (3). Aurora B is a chromosomal passenger protein and essential for chromosome condensation, kinetochore functions, spindle checkpoint activation and cytokinesis completion (4). Aurora C is heavily expressed in testis and is there involved in spermatogenesis, but is also expressed in many cell lines and has been less well studied to date (5). Aurora A is first associated with centrosomes and then with spindle microtubules whereas aurora B localizes to the spinal midzone and finally accumulates at the midbody. MCA-5A12 was raised against full length recombinant human Aurora A expressed in and purified from E. coli
. The antibody was tested for binding to expressed human Aurora A, B and C and shown to react with both Aurora A and B, but not C (see Blot image). The HGNC
names for Aurora A, B and C are AURKA
. Since this antibody cross-reacted between Aurora A and B, we now have in progress monoclonal antibodies specific for only one Aurora gene product which will be posted on our website soon.
1. Glover DM, Leibowitz MH, McLean DA, Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81:95-105 (1995).
2: Hochegger H, Hegarat N, Pereira-Leal JB. Aurora at the pole and equator: overlapping functions of Aurora kinases in the mitotic spindle. Open Biol. Mar 20;3(3):120185 (2013).
3. Barr AR, Gergely F. Aurora-A: the maker and breaker of spindle poles. J Cell Sci.120:2987-96 (2007).
4. Andrew PD, Knatko E, Moore WJ, Swedlow JR. Mitotic mechanics: the auroras come into view. Curr Opin Cell Biol.15(6):672-83 (2003).
5. Tang CJ, Lin CY, Tang TK. Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis. Dev Biol. Dev Biol. 290(2):398-410 (2006).
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