Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human CAD aa 1-100 (N terminal). Database link: P27708
WB: HeLa and HEK293 cell lysate.
ICC/IF: HeLa cells.
Flow Cyt: HeLa cells.
IP: HeLa cell lysate.
This product is a recombinant rabbit monoclonal antibody. Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/100 - 1/300.
1/1000. Predicted molecular weight: 243 kDa.
1/30 - 1/50.
相关性Carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD) is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. This trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the 6-step pathway of pyrimidine biosynthesis is the rate-limiting step in the de novo pyrimidine synthetic pathway. Although most of the CAD protein in the cell is cytosolic, phosphorylation at threonine 456 localizes the protein to the nucleus. While MAPK and EGF phosphorylate CAD at threonine 456, MAPK and c-myc have been found to induce over-expression of CAD.
Immunofluorescence staining of HeLa cells with purified ab40800 at a working dilution of 1/300, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40800 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.
Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling CAD (red) with ab40800 at a 1/120 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.
ab40800 (purified) at 1/30 immunoprecipitating CAD in 10 μg HeLa (Lanes 1 and 2, observed at 250 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP (ab131366) was used as the secondary antibody (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST
Western blot - Anti-CAD antibody [EP710Y] (ab40800)
Anti-CAD antibody [EP710Y] (ab40800) at 1/1000 dilution (purified) + HeLa cell lysate at 1/20000 dilution
Secondary HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution
DeVito SR et al. Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP-sugar synthesis to support viral protein glycosylation. Proc Natl Acad Sci U S A111:18019-24 (2014).
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Alfonso-Pérez T et al. Cytoplasmic interaction of the tumour suppressor protein hSNF5 with dynamin-2 controls endocytosis. OncogeneN/A:N/A (2013).
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