Analysis of Signal Transduction by Phospho Flow Cytometry (CAT#: STEM-CBT-0069-WXH)

Introduction

When a cell needs to convey a signal from receptors on the cell surface, or in response to conditions within the cell itself, post-translational modifications (PTMs) are often a key method of perpetuating that signal. The purpose of PTMs is generally to alter the activity, stability, or function of a protein, and principle among the PTMs is phosphorylation. Phosphorylation is the most well-studied modification used in cell signaling cascades. <br />Phospho-specific flow cytometry is an approach for analyzing and interpreting posttranslational modifications at the single-cell level. This innovation enables the simultaneous and high-throughput examination of two or more phospho-epitopes within multiple cell populations

Add to Cart Please Inquire

Principle Applications Procedure Materials Notes Related Products

Principle

Phospho-specific antibodies are designed to bind to a protein only when that protein is phosphorylated at a specific amino acid. If the protein is only functional when phosphorylated at that amino acid, then the amount of antibody binding becomes a direct readout of the functional activity of that protein within the cell.

Applications

• Investigate multiple activated proteins at the same time.
• Screen for compounds that either enhance or inhibit certain signaling pathways.
• Study Signaling in Human PBMCs(human pertipheral blood mononuclear cells) and other cells.
• Diagnostic testing.
• Statistical mapping of signaling networks.
• High-throughput drug screening.

Procedure

1.Isolation, preparation, and stimulation of cells of interest
2.Fixation and permeabilization
3.staining for surface antigens and phospho-proteins
4.multiparameter flow cytometry analysis.

Materials

1. Phospho-specific antibodies: according to the type of cell to be detected
2. Fluorophore conjugated with
phospho-specific antibody

Notes

1.Requires a protocol that enables the antibody to cross the plasma membrane and reach targets within the cell;
2.The cells must be fixed in order to stop biological activity and prevent the levels of phosphorylation from changing over the course of an experiment.

Other recommended products

Analysis of Proteins Interactions by Flow Cytometry with FRET (CAT#: STEM-CBT-0065-WXH)
Analysis of Cell Viability by Flow Cytometry (CAT#: STEM-CBT-0057-WXH)
Measurement of Intracellular pH by Flow Cytometry (CAT#: STEM-CBT-0056-WXH)
Analysis of Autophagy by Flow Cytometry (CAT#: STEM-CBT-0059-WXH)
Multicolour Flow Cytometry (CAT#: STEM-CBT-0050-WXH)
Immunophenoptyping by Flow Cytometry (CAT#: STEM-CBT-0063-WXH)
Analysis of Membrane potential by Flow Cytometry (CAT#: STEM-CBT-0068-WXH)
Cell Counting by Flow Cytometry (CAT#: STEM-CBT-0072-WXH)