Western blot analysis of extracts from JHH-5, HuH-1, and SK-BR-3 cells using CPS1 Antibody.
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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
CPS1 Antibody recognizes endogenous levels of total CPS1 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CPS1 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 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).
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