The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
1/2000 - 1/10000. Detects a band of approximately 91 kDa (predicted molecular weight: 79 kDa).
Use at 2-5 µg/mg of lysate.
1/100 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Substrate-specific adapter of a DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complex required for cell cycle control, DNA damage response and translesion DNA synthesis. The DCX(DTL) complex, also named CRL4(CDT2) complex, mediates the polyubiquitination and subsequent degradation of CDT1 and CDKN1A/p21(CIP1). CDT1 degradation in response to DNA damage is necessary to ensure proper cell cycle regulation of DNA replication. CDKN1A/p21(CIP1) degradation during S phase or following UV irradiation is essential to control replication licensing. Most substrates require their interaction with PCNA for their polyubiquitination: substrates interact with PCNA via their PIP-box, and those containing the 'K+4' motif in the PIP box, recruit the DCX(DTL) complex, leading to their degradation. In undamaged proliferating cells, the DCX(DTL) complex also promotes the 'Lys-164' monoubiquitination of PCNA, thereby being involved in PCNA-dependent translesion DNA synthesis.
Expressed in placenta and testis, very low expression seen in skeletal muscle. Detected in all hematopoietic tissues examined, with highest expression in thymus and bone marrow. A low level detected in the spleen and lymph node, and barely detectable level in the peripheral leukocytes. RA treatment down-regulated the expression in NT2 cell.
Protein modification; protein ubiquitination.
Belongs to the WD repeat cdt2 family. Contains 7 WD repeats.
Expressed in all fetal tissues examined, included brain, lung, liver, and kidney.
Ubiquitinated by the anaphase promoting complex/cyclosome (APC/C). Phosphorylated upon DNA damage, probably by ATM or ATR.
Nucleus. Nucleus membrane. Cytoplasm > cytoskeleton > centrosome. Nuclear matrix-associated protein. Translocates from the interphase nucleus to the metaphase cytoplasm during mitosis.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma (left) and mouse hybridoma tumor (right) tissues labelling CDT2 with ab72264 at 1/1000 (1µg/ml). Detection: DAB.
Western blot - CDT2 antibody (ab72264)
All lanes : Anti-CDT2 antibody (ab72264) at 0.04 µg/ml
Lane 1 : Whole cell lysate from Hela cells at 50 µg Lane 2 : Whole cell lysate from Hela cells at 15 µg Lane 3 : Whole cell lysate from Hela cells at 5 µg Lane 4 : Whole cell lysate from 293T cells at 50 µg
Immunoprecipitation/ Western Blot of CDT2.
Lane 1: ab72264 at 3µg/mg whole cell lysate.
Lane 2: Control IgG.
ab72264 at 1µg/ml for WB.
Whole cell lysate from Hela cells at 1mg for IP, 20% of IP loaded.
Chemiluminescence with an exposure time of 10 seconds.
ICC/IF image of ab72264 stained HeLa cells. The cells were 4% PFA fixed (10mins) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab72264, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Jascur T et al. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) Triggers MSH2 and Cdt2 Protein-dependent Degradation of the Cell Cycle and Mismatch Repair (MMR) Inhibitor Protein p21Waf1/Cip1. J Biol Chem286:29531-9 (2011).
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