FACE™ PI3 Kinase p85

in-cell Western analysis for phospho PI3 Kinase p85


Fast Activated Cell-based ELISA (FACE™) Kits provide a simple, sensitive method for detecting protein phosphorylation directly in the cell, without making extracts or performing electrophoresis and membrane blotting. These 96-well, high-throughput assays are available in both colorimetric and chemiluminescent formats for over 20 different targets (see list at right). For complete details, click the FACE™ Method tab below.

FACE PI3 Kinase p85 Kits provide 96 rxns each of 2 antibodies that enable you to monitor and compare the levels of both phosphorylated and total PI3K p85. The phospho-PI3K p85 antibody preferentially recognizes peptides and proteins containing phospho-tyrosine at the consensus YXXM motif. The total-PI3K p85 antibody recognizes PI3K p85 regardless of its phosphorylation state. Click the PI3K p85 Info tab below for data and more information.

Name Format Cat No. Price  
FACE™ PI3 Kinase p85 1 x 96 rxns 48175 Discontinued
5 x 96 rxns 48675 Discontinued
FACE™ PI3 Kinase p85 Chemi 1 x 96 rxns 48275 Discontinued
5 x 96 rxns 48775 Discontinued
Measurement of phosphorylated PI3 Kinase p85 (YXXM) and total PI3 Kinase p85 using the FACE PI3 Kinase p85 Cell Based ELISA (In Cell Western) method.

Figure 1: Measurement of phosphorylated and total PI3 Kinase.
NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/ml of PDGF for 5 minutes and fixed. Total and phospho PI3K p85 were each assayed in triplicate using the phospho and total PI3K p85 antibodies included in the FACE PI3 Kinase Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-PI3K p85 antibody preferentially recognizes peptides and proteins containing phospho-tyrosine at the consensus YXXM motif. It was raised against a synthetic phospho-(Tyr) p85 binding motif peptide. Some cross-reactivity is observed for peptides that contain phospho-tyrosine followed by methionine. No cross-reactivity is observed with corresponding nonphosphorylated sequences or with other phospho-tyrosine-containing motifs. The total-PI3K p85 antibody recognizes the total level of PI3K p85 proteins regardless of the phosphorylation state.

PI3 Kinase p85 Overview

Phosphoinositide 3 Kinase (PI3 Kinase, PI3K) is a regulator of cell proliferation, survival and growth. PI3 Kinase catalyzes the production of phosphatidylinositol-3,4,5-triphosphate by phosphorylating phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-biphosphate (PIP2). Growth factors and hormones are responsible for triggering this phosphorylation event and PH domain-containing signaling molecules, including various kinases and their regulators, recognize the resulting product. This activates corresponding cell signaling pathways, eventually leading to cell growth, cell cycle entry, cell migration and cell survival.

FACE™ の使用法

96ウェルプレートで細胞を培養し, 目的の経路を誘導する刺激を与えます。刺激を与えた後, 細胞を固定することでリン酸化を含むタンパク質修飾が保持されます。ブロッキング反応の後, それぞれのウェルに目的タンパク質に特異的な一次抗体, 続いてHRP標識二次抗体を加えます。その後, 発色試薬を加えることにより, 比色定量または化学発光による定量を簡便に行うことができます (図1)。また, キット付属のCrystal Violetを用いて細胞数を補正することができます。それぞれのFACE Kitには目的タンパク質全体とリン酸化型を認識する一次抗体が含まれます。これにより, 細胞数と細胞中の目的タンパク質の総量に対してのリン酸化型タンパク質量を解析することができます。FACEはリン酸化型タンパク質を解析することのできるキットで, 定量的かつ再現性のある結果が得られます。

Flow chart of the FACE in cell Western method that uses a cell based ELISA to measure the levels of the native and phospho forms of signaling proteins and kinases that are activated by phosphorylation.

図1: FACE の操作法

Contents & Storage

Two (or ten) 96-well plates for culturing cells, 96 (or 5 x 96) rxns each of two primary antibodies (1 phospho-specific, 1 specific for native protein), HRP-conjugated secondary antibody, Quenching Solution, 1X Antibody Blocking Buffer, 1X Antibody Dilution Buffer, 10X PBS, 10% Triton X-100, 1% SDS Solution, Developing and Stop Solutions, and Crystal Violet Cell Quantification Solution. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.