|H M R Mk B||Endogenous||16, 18||Rabbit IgG|
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 datasheet or 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 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 October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
NME1/NDKA (D18F10) Rabbit mAb detects endogenous levels of total NME1/NDKA protein. This antibody is predicted to cross-react with NME2/NDKB protein.
Human, Mouse, Rat, Monkey, Bovine
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Pro96 of human NME1/NDKA protein.
The NDK/NME/NM23 kinase family (encoded by the NME gene family) consists of at least eight distinct proteins that exhibit different cellular localization (1). Members of this group inhibit metastasis in a variety of tumor cell types (2). All NDK/NME/NM23 proteins possess nucleoside diphosphatase kinase (NDK) activity and catalyze the phosphorylation of nucleoside diphosphate to the corresponding nucleoside triphosphate to regulate a diverse array of cellular events (3). At least four classes of NDK biochemical activities have been described, including protein-protein interactions (4-6), regulation of GTP-binding protein function (7-9), DNA-associated activities (10,11), and histidine-dependent protein phosphotransferase activity (12). NDK/NME proteins participate in the regulation of a broad spectrum of cellular responses, including development, differentiation, proliferation, endocytosis, and apoptosis (13). Because of its role in metastasis suppression and oncogenesis, NDKA (NME1/NM23-H1) has been widely studied (14). NDKA (NM23-H1) and NDKB (NM23-H2) are encoded by adjacent NME1 and NME2 genes and share 90% sequence identity. Two serine residues (Ser122 and Ser144) on NDKA/NM23-H1 can be phosphorylated by AMPKα1, but only phosphorylation at Ser122 determines whether NDKA channels ATP to AMPKα1. This regulates AMPKα1 activity towards ACC1, an important regulator of fatty acid metabolism (15). Mutation of NDKB/NM23-H2 at Ser122 (S122P) in melanoma cells results in altered phosphoryl transfer activity (16).
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