Western blot analysis of extracts from various cell lines using NNMT Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Expression levels of NNMT among cell lines are consistent with expectations based on publicly available bioinformatic databases.
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
NNMT Antibody recognizes endogenous levels of total NNMT protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu12 of human NNMT protein. Antibodies are purified by protein A and peptide affinity chromatography.
Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme expressed primarily in liver and adipose tissue. It catalyzes the transfer of a methyl group from S-Adenosyl-methionine (SAM) to nicotinamide, yielding 1-methylnicotinamide (MNAM) and S-Adenosyl-L-homocysteine (SAH) (1). This N-methylation enzymatic activity plays an important role in the biotransformation of drugs and xenobiotics, and also contributes to the metabolism of vitamin B3 (2). Knockdown of NNMT was shown to increase both SAM and NAD+ levels in white adipose tissue of high-fat diet-fed mice, resulting in increased energy expenditure and protection against diet-induced obesity (3). In contrast, increased liver NNMT expression in humans and mice correlated with an improved metabolic profile, through MNAM-mediated SIRT1 protein stabilization (4). In cancer cells, overexpression of NNMT resulted in excess consumption of methyl units from SAM, leading to histone hypomethylation that substantially altered the epigenetic landscape (5). These and other research studies have suggested that NNMT expression may have utility as a diagnostic and prognostic biomarker in cancer (6-8).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.