Isoform specificity of 14-3-3γ (D15B7) Rabbit mAb. Recombinant, purified, GST-tagged 14-3-3 protein isofoms (2 µg each)were resolved by SDS-PAGE, transferred to nitrocellulose and blotted with either 14-3-3 γ (D15B7) Rabbit mAb (upper) or GST (91G1) Rabbit mAb #2625 (lower).
Western blot analysis of extracts from various cell lines using 14-3-3 γ (D15B7) 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 nonfat dry milk, 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.
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: 263
14-3-3γ (D15B7) Rabbit mAb recognizes endogenous levels of total 14-3-3 γ protein. This antibody does not cross-react with other mammalian 14-3-3 isoforms and is predicted to detect 14-3-3γ orthologs in other species, including frog and fish.
Human, Mouse, Rat, Monkey, Pig
Chicken, Xenopus, Zebrafish
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile79 of human 14-3-3 γ protein.
The 14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic and nutrient-sensing pathways (1,2). 14-3-3 proteins are highly conserved and ubiquitously expressed. There are at least seven isoforms, β, γ, ε, σ, ζ, τ, and η that have been identified in mammals. The initially described α and δ isoforms are confirmed to be phosphorylated forms of β and ζ, respectively (3). Through their amino-terminal α helical region, 14-3-3 proteins form homo- or heterodimers that interact with a wide variety of proteins: transcription factors, metabolic enzymes, cytoskeletal proteins, kinases, phosphatases, and other signaling molecules (3,4). The interaction of 14-3-3 proteins with their targets is primarily through a phospho-Ser/Thr motif. However, binding to divergent phospho-Ser/Thr motifs, as well as phosphorylation independent interactions has been observed (4). 14-3-3 binding masks specific sequences of the target protein, and therefore, modulates target protein localization, phosphorylation state, stability, and molecular interactions (1-4). 14-3-3 proteins may also induce target protein conformational changes that modify target protein function (4,5). Distinct temporal and spatial expression patterns of 14-3-3 isoforms have been observed in development and in acute response to extracellular signals and drugs, suggesting that 14-3-3 isoforms may perform different functions despite their sequence similarities (4). Several studies suggest that 14-3-3 isoforms are differentially regulated in cancer and neurological syndromes (2,3).
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