AlphaLISA SureFire Ultra Human and Mouse Total Src Detection Kit, 10,000 Assay Points

Total-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.

Activation of Src levels in H₂O₂ treated A549 cells

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with 25 mM H₂O₂ for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr419) and Total Src levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, H₂O₂ triggered an increase in the levels of Phospho Src (Tyr419) while Total Src remained unchanged.

Src Total expression in various cell lines

Adherent cells were grown to confluency in a T175 flask in complete medium and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested then washed in HBSS and lysed with Lysis Buffer at a density of 1.6 x 10⁶ cells/mL.

Src levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Src expression was detected in a wide range of human and mouse cell lines.

Dilutions of recombinant Src (Abcam, ab79635) were prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

The dotted line represents assay background.

Cell lysate was prepared from SH-SY5Y cells seeded in T175 flasks and cultured to confluence. Cells were lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Src Phospho (Tyr419) and Total levels were evaluated using by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells is indicated. The dotted line represents assay background. The Src assay can detect Src expression in less than 400 cells.