Phospho-c-Src (Tyr419) and Total c-Src Assay Kits

Phospho-c-Src (Tyr419) and Total c-Src assays to identify selective c-Src kinase inhibitors for cancer treatment

These cell-based assays are designed to monitor the expression level and the autophosphorylation of c-Src on Tyr419, which is a hallmark of its activation.

c-Src is a member of the Src kinase family and shares a high degree of sequence identity with the ten other members (Fyn, Lyn, Lck, Frk, Blk, Hck, Yrk, Fgr, Srm and Yes).
This protein is a membrane-associated, non-receptor tyrosine kinase. Its autophosphorylation on the residue Tyr419 signs its full activation.

c-Src kinase is derived from the proto-oncogene c-Src and regulates cell proliferation, differentiation, survival, morphology, adhesion and migration. It is over-expressed and highly activated in many human cancers (e.g. breast, colon and pancreatic), making it a promising target for cancer therapy.

   Check our lysis buffer compatibility

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total c-Src assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-c-Src antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with c-Src, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

Assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-c-Src can be quantitatively detected using the HTRF phospho-c-Src (Tyr419) and total c-Src cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

For added flexibility, the assays can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred into a 384-well low volume white microplate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

Product performances

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

Human HEK293 embryonic kidney cells were plated at 30,000 cells/well in a 96-well plate and incubated for 24h at 37°C, 5% CO2. The following day, the cells were transfected with a c-Src siRNA or with a negative control siRNA for 48h (with medium renewal after 24h). The cells were then treated with increasing concentrations of H2O2 for 10 minutes before medium removal and lysis 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-c-Src or total c-Src detection reagents were added. The HTRF signal was recorded after 4 hours of incubation.

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 both HTRF® phospho- and total c-Src assays are selective for c-Src and do not cross-react with other Src family members.

2. HTRF assays compared to Western Blot using phospho- & total c-Src cellular assays on human A431 cancer cells

Human A431 epidermoid carcinoma cells were seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 80% confluency was reached. After treatment with 10 mM H2O2 for 10 minutes, the cells were lysed with 3 mL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF® phospho or total detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.

Using HTRF® Phospho-c-Src, just 620 cells were sufficient for minimal signal detection whereas 20,000 cells were needed for a Western Blot signal. The HTRF® phospho-c-Src assay is 32-fold more sensitive than the Western Blot. Using HTRF® Total c-Src, only 310 cells were sufficient for minimal signal detection, while 2,500 cells were needed for a Western Blot signal, demonstrating that the HTRF® total c-Src assay is 8-fold more sensitive than the Western Blot.

Image of the comparison between Western blot and HTRF using phospho- and total c-Src on human A431 cells

Image of the comparison between Western blot and HTRF using phospho- and total c-Src on human A431 cells

3. Inhibition of c-Src activity in human HEK293 embryonic kidney cells using the Src kinase inhibitor Bosutinib

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 minutes 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.

4. 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.

5. Validation on other human cancer cell lines

Culture-treated 96-well plates were seeded with human HepG2 hepatocellular carcinoma cells (100K cells/well), human HeLa cervical carcinoma cells (100K cells/well) and human MCF-7 breast cancer cells (25K cells/well). After incubation for 24h at 37°C, 5% CO2, the cells were treated or not with 20 mM H2O2 for 10 minutes. The medium was then 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.

6. Cell density experiment on mouse NIH/3T3 cells treated with H2O2

Mouse NIH/3T3 embryonic fibroblast cells were plated at different cell densities in a 96-well plate and incubated for 24h at 37°C, 5% CO2. The following day, the cells were treated with increasing concentrations of H2O2 for 10 minutes. The medium was then 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.

7. Correlation with a commercial ELISA kit on human HEK293 cells treated with Bosutinib

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 minutes with increasing concentrations of Bosutinib and then stimulation with 20 mM H2O2 for 10 minutes, the medium was removed and the cells were lysed either with 50 µL of HTRF supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, or with 50 µL of the ELISA lysis buffer following manufacturer’s instructions. For HTRF detection, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF® phospho-c-Src detection reagents were added. The HTRF signal was recorded after 4 hours of incubation. For colorimetric ELISA detection, 100 µL of lysate were transferred into the ELISA plate and the next steps were carried out according to manufacturer’s instructions before measuring OD at 450 nm.

Similar IC50 values for Bosutinib were obtained using the two methods, demonstrating that HTRF is well correlated to ELISA.

Simplified pathway

c-Src, a key signaling kinase in cancer

c-Src is a member of the Src kinase family which includes ten other members (Fyn, Lyn, Lck, Frk, Blk, Hck, Yrk, Fgr, Srm and Yes).
This membrane-associated, non-receptor protein tyrosine kinase is ubiquitously expressed in all cell types.
The kinase is inactive when phosphorylated on Tyr530 by C-terminal Src kinase (CSK). Conversely, 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 (ERK1/2, JNK & p38), STATs and NFκB. 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
c-Src total kit - 500 tests64NSRPEG
-
c-Src total kit - 10,000 tests64NSRPEH
-
c-Src phospho-Y419 kit - 500 tests64SRCPEG
-
c-Src phospho-Y419 kit - 10,000 tests64SRCPEH
-

Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein lysis buffer #3 - 130 mL64KL3FDF
-
c-Src total kit control lysate64NSRTDA
-
-
c-Src phospho-Y419 kit control lysate64SRCTDA
-
-

ドキュメント