AlphaLISA SureFire Ultra Human and Mouse Total HRAS Detection Kit, 500 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.

Specificity of HRAS Total assay

Specificity of the HRAS Total assay was assessed by evaluating recombinant HRAS, NRAS and KRAS proteins.

Dilutions of NRAS (MyBioSource, MBS143929), HRAS (Abcam, ab61239) and KRAS (Abcam, ab156968) proteins were prepared in Lysis Buffer at the indicated concentrations. HRAS Total levels were 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.

HRAS Total expression was only detected with HRAS recombinant protein, while no cross-reactivity was observed with NRAS and KRAS proteins. These results demonstrate the specificity of the HRAS Total assay as these three proteins shares approximately 86% sequence identity. Dotted line represents assay background.

Knockout validation of HRAS Total Assay

HRAS Total protein levels were assessed in HEK293 wild type (WT) and HRAS KO (Abcam, ab260858) cell lines cultured to confluency in T175 flasks.

Each flask was lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and evaluated for HRAS Total using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was 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.

HRAS Total was only detected in WT cells, demonstrating assay specificity. Dotted line represents assay background.

HRAS expression in various cell lines

Adherent cells were grown to confluency in T175 flasks at 37°C, 5% CO₂, and were lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

HRAS Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (5,000 adherent cells 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.

HRAS is expressed in a wide variety of cell lines. Very high levels of expression were detected in PC3, A549 and A431 cells.

Assay Sensitivity - Cell lysate dilution

Cell lysate was prepared from HeLa cells cultured to confluence a T175 flask and lysed with Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and HRAS Total levels were evaluated using the AlphaLISA SureFire Ultra assay. 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 per datapoint is indicated. The dotted line represents assay background. The assay can detect HRAS Total in less than 500 cells/datapoint.