FlowCellect™ Src Activation Dual Detection Kit

FlowCellect™ Src Activation Dual Detection Kit

FlowCellect

25 Tests / Kit

Millipore’s FlowCellect™ Src Activation Dual Detection kit includes two directly conjugated antibodies, a phospho-specific Anti-phospho-Src (Tyr416)-Alexa Fluor® 488 and an Anti-Src-Alexa Fluor® 647 conjugated antibody to measure total levels of Src. This two color flow cytometry kit is designed to detect the extent of Src pathway activation by measuring the Src phosphorylation relative to the total Src 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 Src activation after stimulation. Moreover, simultaneous measurement of both total and phospho-Src 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.

Src pathway activation in response to receptor tyrosine kinase stimuli (e.g. EGFR stimulated by EGF) plays a significant role in the regulation of essential cellular processes. Dysregulation of Src signaling makes Src a key molecule in evaluation of tumor progression as it can provide oncogenic signals for cell survival, mitogenesis, angiogenesis, and metastasis. As a result, newer generations of Src inhibitors are being actively investigated as cancer therapeutics. When stimulating A431 cells with EGF for 5 minutes the total protein level remains constant compared to unstimulated cells, while phospho-Src levels are increased in all cells indicating that there is no competition between the two antibodies for their target epitopes.

Millipore’s FlowCellect™ Activation Dual Detection kits are a series of flow cytometry products which include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and, by doing so, eliminate false positives while enhancing the signal to noise ratio.

Src is a non-receptor tyrosine kinase involved in many key signaling cascades which has been implicated to play a major role in tumorigenesis. Elevated Src expression has been seen in multiple solid tumors including breast cancer. In fact, Src kinase activity is greatly increased in breast cancer tissue compared with normal breast tissue. Src plays a role in signaling and cross talk between growth-promoting pathways, such as ER and EGFR family signaling pathways, known to be active in breast cancer.

Because of its essential role in many intracellular signaling pathways, interrupting Src signaling may disrupt oncogenic pathways. Src physically interacts with activated receptor tyrosine kinases (RTKs) and creates a positive-regulatory loop that contributes to the robustness and persistence of RTK signaling. Over-expression of RTKs due to their amplification or mutation is a very common occurrence in solid tumors, suggesting that a dysregulated interplay between RTKs and Src may effect an abnormally elevated Src activity. Src can associate with the over-expressed EGFR to cause synergistic mitogenicity, which can represent one mechanism by which Src contributes to the growth of tumor cells. In a high percentage of human neoplasms such as colorectal, breast, prostate, pancreas, head and neck, and lung carcinoma, to name a few, Src is over-expressed or hyperactivated, thus, making its dysregulation a major oncogenic signature found in cancer.

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.

Lot to lot in-process testing on all kit components

• 1. 20X Anti-phospho-Src (Tyr416) Alexa Fluor® 488: (Part No. CS206379) One vial containing 150 µL.
• 2. 20X Anti-Src-Alexa Fluor® 647: (Part No. CS206380) 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

4-8°C for antibodies and buffers

Fluorescent

FlowCellect™ Src Activation Dual Detection Kit

Flow Cellect

ExpressPro & inCyte

Monoclonal Antibody

Research