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HTRF Human and Mouse Total c-Src Detection Kit, 500 Assay Points

The Total c-Src kit monitors cellular c-Src expression level and can be used as a normalization assay for the phospho-c-Src kit.

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

The Total c-Src kit monitors cellular c-Src expression level and can be used as a normalization assay for the phospho-c-Src kit.

View product information

Product variants

Unit Size: 500 assay points

Part #:

64NSRPEG

Unit Size: 10,000 assay points

Part #:

64NSRPEH

Unit Size: 50,000 assay points

Part #:

64NSRPEY

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

Overview

The Total c-Src cellular assay monitors total c-Src and is used as a normalization assay with the phospho-c-Src kit. This protein is a member of the Src kinase family and shares a high degree of sequence identity with the ten other members. This protein is a membrane-associated, non-receptor tyrosine kinase. Its autophosphorylation on the residue Tyr419 indicates its full activation. c-Src kinase is over-expressed and highly activated in many human cancers, making it a promising target for cancer therapy.

How it works

Total-c-Src assay principle

The Total-c-Src assay quantifies the expression level of c-Src in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-c-Src assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of c-Src in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.

Total-c-Src 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total-c-Src HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-c-Src 1-plate assay protocol

Detection of total cSrc with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Assay validation

Selectivity of HTRF assays for c-Src validated by siRNA experiments on human HEK293 cells

Human HEK293 embryonic kidney cells were plated and cultured for 24h. Cells were then transfected with a c-Src siRNA or with a negative control and treated with increasing concentrations of H2O2 before medium removal and lysis. Lysates were transferred into a 384-well low volume white microplate and kit reagents were added. Cell treatment with the c-Src siRNA led to a huge decrease of the HTRF signal (~ 75%) compared to the cells transfected with the negative control siRNA, demonstrating that this kits is selective for c-Src and do not cross-react with other Src family members.

HTRF vs WB using total c-Src assays on A431 cancer cells

Human A431 cancer cells were seeded in a T175 flask and cultured until 80% confluency was reached. Following cell lysis, soluble supernatants were collected via centrifugation and serial dilutions were performed in the supplemented lysis buffer and transferred into a 384-well low volume white microplate before finally adding HTRF total detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF. The HTRF total c-Src assay is 8-fold more sensitive than WB.

Bosutinib Inhibition of c-Src activity in HEK293 cells

HEK293 cells were plated at 100,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 30 minutess with increasing concentrations of Bosutinib and then stimulation with 20 mM H2O2 for 10 minutes, the medium was removed and the cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho- or total c-Src detection reagents were added. The HTRF signal was recorded after 4 hours of incubation.

c-Src activation in human A431 epidermoid carcinoma cells using H2O2

A431 cells were plated at 100,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 10 minutes with increasing concentrations of H2O2, the medium was removed and the cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho- or total c-Src detection reagents were added. The HTRF signal was recorded after 4 hours of incubation.

Validation on other human cancer cell lines

Culture-treated 96-well plates were seeded with human HepG2 hepatocellular carcinoma cells, human HeLa cervical carcinoma cells and human MCF-7 breast cancer cells. After incubation, cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutess at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF phospho- or total c-Src detection reagents were added. The HTRF signal was recorded after 4 hours of incubation.

Simplified pathway

c-Srt simplified pathway

c-Src is a membrane-associated, non-receptor protein tyrosine kinase that's ubiquitously expressed in all cell types. The kinase is inactive when phosphorylated on Tyr530. Dephosphorylation of Tyr530 followed by autophosphorylation on Tyr419 is a hallmark of its full activation. c-Src is mainly activated by RTKs, integrins and ROS, and is a key signaling intermediary regulating numerous pathways such as AKT, MAPKs, STATs and NF?B. It regulates cell proliferation, differentiation, survival, morphology, adhesion and migration. Precise regulation of Src activity is therefore critical for normal cell growth. This derived-proto-oncogene is over-expressed and highly activated in many human cancers (e.g. breast, colon and pancreatic), making it a promising target for cancer therapy.

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Oncology & Inflammation
Unit Size	500 assay points