For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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: 263
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CPS1/Hep Par-1 protein. Antibodies are purified by peptide affinity chromatography.
Carbamoyl phosphate synthetase 1 (CPS1), a rate-limiting enzyme in the urea cycle, catalyzes the conversion of ammonia and bicarbonate to carbamoyl phosphate in mitochondria. Non-small cell lung carcinoma (NSCLC) cells with oncogenic KRAS and loss of the tumor suppressor LKB1 express CPS1 and depend on this enzyme for growth. CPS1 maintains pyrimidine/purine balance in these cancer cells. Silencing CPS1 reduces the pyrimidine to purine ratio and stalls DNA synthesis, leading to DNA damage and cancer cell death (1). In addition, the tumor suppressor p53 represses the expression of urea cycle enzymes CPS1, OTC, and ARG1 and, therefore, causes the accumulation of ammonia, suppressing cancer growth (2). Furthermore, research studies suggest that hypermethylation-mediated downregulation of CPS1 expression may contribute to the progression of normal hepatocytes to hepatocellular carcinoma (HCC) (3). CPS1 was identified to be the antigen detected by Hep Par-1 antibody (4).
UniProt ID: P31327
Entrez-Gene Id: 1373
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