FlowCellect™ PI3K Activation Dual Detection Kit

FlowCellect™ PI3K Activation Dual Detection Kit

FlowCellect

25 Tests / Kit

Millipore’s FlowCellect™ PI3K Activation Dual Detection kit includes two directly conjugated antibodies, a phospho-specific Anti-phospho-Akt (Ser473)-Alexa Fluor® 488 and an Anti-Akt-Alexa Fluor® 647 conjugated antibody to measure total levels of Akt. This two color flow cytometry kit is designed to measure the extent of Akt phosphorylation relative to the total Akt expression in any given cell population. By doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of Akt activation after stimulation. Moreover, simultaneous measurement of both total and phospho-Akt confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho:total ratio within a mixed population.

All FlowCellect kits are supplied with optimized fixation, permeabilization, wash, and assay buffers to provide researchers the ability to detect the activation of Akt in flow applications. Sufficient reagents are provided for 25 two-color samples. The kit includes all optimized fluorescently labeled antibodies and buffers necessary for cell preparation and analysis.

Akt/PKB is a Ser/Thr kinase and a major known effecter of the PI3 Kinase pathway. It is involved in multiple signaling pathways that relate to many biological processes including metabolism, apoptosis, cell cycle control, angiogenesis, differentiation, and cell growth and proliferation. In mammals, three isoforms of Akt (Akt1/PKCα, Akt2/PKBβ, and Akt3/PKBγ) exists. They exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites. Akt1 is the predominant isoform that is in most tissues and is thought to have a dominant role in growth, survival, embryonic development, and adipocyte differentiation. Akt2 is correlated with the regulation of glucose homeostasis and is the predominant isoform expressed in insulin-responsive tissues. Akt3 is abundant in brain tissue. Each Akt isoform is composed of three functionally distinct regions: an N-terminal Pleckstrin Homology (PH) domain that provides a lipid-binding module, a central catalytic domain containing Thr308, and a C-terminal hydrophobic motif containing Ser473. The activation of AKT is dependent on a dual regulatory mechanism that requires both its translocation to the plasma membrane and dual phosphorylation on Thr308 and Ser473 by PDK1 and the TORC2 complex, respectively.

Phosphorylation of Akt at Ser473 serves as a major signaling node to activate its many downstream effectors such as mTOR, Bad, GSK3, Forkhead, and Caspases, leading to multiple biological processes such as cell survival, cell growth, and cell metabolism.

Flow Cytometry

Human

• Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
• Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

4-8°C for antibodies and buffers

FlowCellect™ PI3K Activation Dual Detection Kit

Flow Cellect

ExpressPro

Monoclonal Antibody

Research

Lot to lot in-process testing on all kit components

• 1. 20X Anti-phospho-Akt (Ser473) Alexa Fluor® 488 Conjugated Antibody: (Part No. CS203310) One vial containing 150 µL.
• 2. 20X Anti-Akt/PKB-Alexa Fluor® 647 Conjugated Antibody: (Part No. CS205073) One vial containing 150 µL.
• 3. Fixation Buffer: (Part No. CS202122) One bottle containing 13 mL.
• 4. 10X Wash Buffer: (Part No. CS202123) One bottle containing 13 mL.
• 5. 5X Assay Buffer: (Part No. CS202124) One 55 mL bottle
• 6. 1X Permeabilization Buffer: (Part No. CS203284) One bottle containing 13 mL.

Mouse

Fluorescent