Western blot analysis of extracts from various cell lines using NAE1/APPBP1 (D9I4Z) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human NAE1/APPB1 protein (hNAE1-Myc/DDK; +), using NAE1/APPBP1 (D9I4Z) Rabbit mAb.
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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.
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
NAE1/APPBP1 (D9I4Z) Rabbit mAb recognizes endogenous levels of total NAE1/APPBP1 protein. This antibody does not cross-react with E1 activating enzymes for either ubiquitin or other ubiquitin-like proteins.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala179 of human NAE1/APPBP1 protein.
Similar to ubiquitin, NEDD8 is covalently linked to target proteins through an enzymatic cascade composed of NEDD8-specific E1 (activating)- and E2 (conjugating)-enzymes (1,2). The E2 ligase specific for NEDD8 is Ubc12 (3-5). Ubc12 forms a heterodimeric conjugate with NEDD8 in order to catalyze the transfer of NEDD8 from E1 to lysine side chains of target proteins (1,2). Well known targets of NEDD8 are cullin-based RING E3 ligases. Neddylation of cullin isoforms activates the related ubiquitin E3 complex by promoting its interaction with a cognate ubiquitin-E2 ligase (6-7). Neddylation of Cul-1 complexes containing βTrCP and SKP2 has been shown to be required for controlling the stability of important signaling targets such as IκB, NF-κB, and p27 Kip (8-10), thereby regulating cell cycle progression, signaling cascades, and developmental programming processes (11).
NAE1/APPBP1 (NEDD8-Activating Enzyme 1/Amyloid Beta Precursor Protein Binding Protein 1) exists in a heterodimeric complex with UBA3. This complex functions as an E1 NEDD8-activating enzyme, which utilizes ATP to adenylate the C-terminal glycine of NEDD8 (12-14). Research studies suggest that inhibition of the APPBP1-UBA3 complex may be of therapeutic value for the treatment of human cancers (15,16).
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