AlphaLISA SureFire Ultra Human Total EGF Receptor E746A-750 Deletion Detection Kit, 100 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.

EGF Receptor E746-A750 expression in various cell lines

The presence of the EGF Receptor E746-A750 deletion mutation was evaluated in various EGFR expressing cell lines using the AlphaLISA SureFire Ultra assay kit. Cells were grown to confluency in a T175 flask and lysed with Lysis Buffer at a density of 150,000 cells/mL. For the detection step, 10 µL of lysate (approximately 15,000 cells) 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.

As expected, EGF Receptor E746-A750 deletion mutation was only present in HCC-827 cells.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HCC827 seeded in T175 flasks and cultured to confluence. Cells were lysed in 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and assayed for EGF Receptor E746-A750 deletion levels using the AlphaLISA SureFire Ultra kit. 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/datapoint is indicated. The dotted line represents assay background. The assay can detect Total EGF Receptor E746-A750 deletion expression down to 150 cells.