Western blot analysis was performed on whole cell extracts (30 ug lysate) of HeLa (Lane 1), MDA-MB-231 (Lane 2), HEK-293 (Lane 3), A549 (Lane 4), U-87 MG (Lane 5), T98G (Lane 6), DU 145 (Lane 7), Jurkat (Lane 8) and Caco-2 (Lane 9).The blots were probed with AKT3 Rabbit polyclonal Antibody (Product# PA1-41700, 2 ug/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 ug/ml, 1:2500 dilution). A 56 kDa band corresponding to AKT3 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to residues 119-136 of human Akt3.|
|Storage buffer||PBS with 0.05% BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Suggested positive control: Hela whole cell extract, HeLa, antigen standard for AKT3 (transient overexpression lysate).
Akt, protein kinase B (PKB), is a serine/threonine kinase which is involved in many cellular signaling pathways and acts as a transducer of many functions initiated by growth factor receptors that activate phosphtidylinositol 3-kinase (PI 3-kinase). The major activity of Akt/PKB is to mediate cell survival. Akt/PKB is also believed to be a critical factor in the genesis of cancer as the tumor suppressor PTEN was found to antagonise PI-3 kinase and Akt/PKB kinase activity. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1, 2 and 3 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. Akt/PKB phosphorylation is critical for its activity.
IP-MS enrichment of AKT3 (LFQ intensity): AKT3 was enriched 145-fold from HEK293 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Part No. 90409) and AKT3 antibody (Part No. PA1-41700). The STRING database (www.string-db.org) was used to identify the protein interactor list. See more information on IP-MS verification of antibody selectivity. IP-MS validation info.
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