Western blot analysis of extracts from various cell lines using CDCA2 (D7T4P) Rabbit mAb.
Western blot analysis of ACHN cell extracts, untreated (-) or synchronized in mitosis by treatment with Nocodazole #2190 (10 nM, 16 hr; +), using CDCA2 (D7T4P) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
CDCA2 (D7T4P) Rabbit mAb recognizes endogenous levels of total CDCA2 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr50 of human CDCA2 protein.
Cell division cycle associated 2 (CDCA2, Repo-Man) is a cell-cycle protein that recruits protein phosphatase 1 (PP1) to mitotic chromatin at anaphase onset, which is essential for cell proliferation (1). Carboxy-terminal phosphorylation of CDCA2 at Ser893 by Aurora B inhibits the protein and leads to diffuse localization during prometaphase and metaphase. Dephosphorylation of CDCA2 by PP2A is necessary for CDCA2/PP1 complex reformation (2). The CDCA2/PP1 complex is required for chromatin binding and dephosphorylation of histone H3 at Thr3, Ser10, and Ser28 (2-4). The CDCA2/PP1 complex is also involved in nuclear envelope reformation during mitotic exit for proper progression through the M/G1 transition (4). The interaction of CDCA2 with importin beta and Nup153, which is required for nuclear envelope formation, is negatively regulated by CDK phosphorylation of the amino-terminal domain of CDCA2 (5). CDCA2 may play a role in DNA repair as the release of CDCA2 from chromatin at sites of DNA damage promotes the activation of DNA damage response (6). These results imply that the CDCA2/PP1 complex may play a part in cancer progression. Research studies indicate that CDCA2 may serve as a prognostic marker, as increased CDCA2 expression is seen in a number of cancers, including melanoma, neuroblastoma tumors, squamous cell carcinoma, and synovial sarcomas (7-9).
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