Western blot analysis of extracts from Ramos and HeLa cells, untreated or treated with TPA (Phorbol-12-Myristate-13-Acetate, 200 nM for 30 min) or λ-phosphatase, using Phospho-Numb (Ser276) Antibody (upper) or Numb (C29G11) Rabbit mAb #2756 (lower).
|REACTIVITY||H M R Mk|
|MW (kDa)||72, 74|
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 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
Phospho-Numb (Ser276) Antibody detects endogenous levels of Numb protein only when phosphorylated on Ser276.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to Ser276 of the human Numb protein. Antibodies are purified by protein A and peptide affinity chromatography.
Numb contains an amino-terminal phosphotyrosine-binding (PTB) domain and carboxy-terminal endocytic binding motifs for α-adaptin and EH (Eps15 homology) domain-containing proteins, indicating a role in endocytosis (1,2). There are four mammalian Numb splicing isoforms that are differentially expressed and may have distinct functions (3-5). Numb acts as a negative regulator of Notch signaling by promoting ubiquitination and degradation of Notch (6). The protein is asymmetrically segregated into one daughter cell during cell division, producing two daughter cells with different responses to Notch signaling and different cell fates (7,8). The localization of Numb can also be regulated by G-protein coupled receptor (GPCR) and PKC signaling (9).
Numb can be phosphorylated at several sites including Ser7, Ser276 and Ser295. Phosphorylation at these sites regulates asymmetric membrane localization of Numb and integrin endocytosis (10-12).
Explore pathways + proteins related to this product.