Western blot analysis of extracts from SW620, MEF, and C6 cells using CAD Antibody.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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 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 June 2020
Protocol Id: 10
CAD Antibody recognizes endogenous levels of total CAD protein. This antibody may cross-react with a protein of unknown origin at ~45 kDa in some cell lines.
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CAD protein. Antibodies are purified by protein A and peptide affinity chromatography.
CAD is essential for the de novo synthesis of pyrimidine nucleotides and possesses the following enzymatic activities: glutamine amidotransferase, carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase. Thus, the enzyme converts glutamine to uridine monophosphate, a common precursor of all pyrimidine bases, and it is necessary for nucleic acid synthesis (1). In resting cells, CAD is localized mainly in the cytoplasm where it carries out pyrimidine synthesis. As proliferating cells enter S phase, MAP Kinase (Erk1/2) phosphorlyates CAD at Thr456, resulting in CAD translocation to the nucleus. As cells exit S phase, CAD is dephosphorylated at Thr456 and phosphorylated at Ser1406 by PKA, returning the pathway to basal activity (2). Various research studies have shown increased expression of CAD in several types of cancer, prompting the development of pharmacological inhibitors such as PALA. Further studies have identified CAD as a potential predictive early marker of prostate cancer relapse (3).
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