Western blot analysis of extracts from HT-29 cells, untreated (-) or synchronized in mitosis by treatment with thymidine (2 mM, 17 hr; +) and Nocodazole #2190 (100 ng/ml, 24 hr; +), using Aurora A (D3E4Q) Rabbit mAb #14475 (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Confocal immunofluorescent analysis of mitotic HeLa cells during metaphase (left) and anaphase (right) using Phospho-Aurora A (Thr288) (C39D8) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of untreated Jurkat cells using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb compared to propidium iodide (DNA content). The boxed population indicates phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)-positive cells.
Flow cytometric analysis of Jurkat cells using Aurora A (D3E4Q) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow cytometric analysis of Hela cells, using Aurora B/AIM1 Antibody (blue) compared to a nonspecifc negative control antibody (red).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from hydroxyurea or nocodazole treated HeLa and HT29 cells using Phospho-Aurora A (Thr288) (C39D8) Rabbit mAb.
Confocal immunofluorescent analysis of HT-1080 cells using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb (green), β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red), and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (blue).
Confocal immunofluorescent analysis of HeLa cells using Aurora A (D3E4Q) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from Hela, NIH/3T3 and C6 cells, untreated or nocodazole-treated (50 ng/ml, 24 hrs), using Aurora B/AIM1 Antibody.
Western blot analysis of extracts from HeLa, L929 and C6 cells, treated with 4 mM hydroxyurea for 20 hours to induce G1/S phase or treated with 100 nM paclitaxel or 100 ng/ml nocodazole for 20 hours to induce G2/M phase, using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D11A13) XP® Rabbit mAb (upper) or Aurora B/AIM1 Antibody #3094 (lower).
Immunoprecipitation of Aurora A from HeLa cell extracts using Rabbit (Da1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Aurora A (D3E4Q) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Aurora A (D3E4Q) Rabbit mAb.
Western blot analysis of extracts from HT-29 cells, untreated (-) or synchronized in mitosis by treatment with thymidine (2 mM, 17 hr; +) and Nocodazole #2190 (100 ng/ml, 24 hr; +), using Aurora A (D3E4Q) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
|Phospho-Aurora A (Thr288) (C39D8) Rabbit mAb 3079||20 µl||
|Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb 2914||20 µl||
||H M R||35, 40, 48||Rabbit IgG|
|Aurora A (D3E4Q) Rabbit mAb 14475||20 µl||
|Aurora B/AIM1 Antibody 3094||20 µl||
||H M R Mk||40||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Aurora Antibody Sampler Kit provides an economical means to investigate the G2/M phase of the cell cycle. The kit contains enough primary and secondary antibodies to perform two western blots with each antibody.
Each antibody in the Aurora Antibody Sampler Kit detects endogenous levels of its respective target protein and does not cross-react with other family members.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide and are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with recombinant human proteins or synthetic peptides.
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, B, and C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Aurora kinase functional influences span from G2 phase to cytokinesis and may be involved in key cell cycle events such as centrosome duplication, chromosome bi-orientation and segregation, cleavage furrow positioning, and ingression (3). Aurora A is detected at the centrosomes, along mitotic spindle microtubules, and in the cytoplasm of mitotically proliferating cells. Aurora A protein levels are low during G1 and S phases and peak during the G2/M phase of the cell cycle. Phosphorylation of Aurora A at Thr288 in its catalytic domain increases kinase activity. Aurora A is involved in centrosome separation, maturation, and spindle assembly and stability. Expression of Aurora B protein also peaks during the G2/M phase of the cell cycle; Aurora B kinase activity peaks at the transition from metaphase to the end of mitosis. Aurora B associates with chromosomes during prophase prior to relocalizing to the spindle at anaphase. Aurora B regulates chromosome segregation through the control of microtubule-kinetochore attachment and cytokinesis. Expression of both Aurora A and Aurora B during the G2/M phase transition is tightly coordinated with histone H3 phosphorylation (4,5); research investigators have observed overexpression of these kinases in a variety of human cancers (2,4). Aurora C localizes to the centrosome from anaphase to cytokinesis and both mRNA and protein levels peak during G2/M phase. Although typical Aurora C expression is limited to the testis, research studies report overexpression of Aurora C is detected in various cancer cell lines (6).
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