AlphaLISA SureFire Ultra Human and Mouse Total NRAS 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 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 NRAS Total assay

Specificity of the NRAS assay for NRAS protein was assessed by evaluating recombinant NRAS, HRAS 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. NRAS Total levels was 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.

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

Knockout validation of NRAS Total Assay

NRAS expression was assessed in A549 wild type (WT) and NRAS KO (Abcam, ab286611) 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 NRAS 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.

NRAS expression was only detected in WT cells, demonstrating assay specificity. Dotted line represents assay background.

NRAS expression in human PBMCs

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll Plaque Plus (Merck GE17-1440-02). PBMCs were lysed at 6 x 10⁶ cells/mL with Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and NRAS levels were evaluated 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 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.

Dotted line represents assay background.

NRAS 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.

NRAS levels were evaluated by using the AlphaLISA SureFire Ultra assay. 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.

NRAS is expressed in a wide variety of cell lines. High levels of expression were detected in MCF7 and THP-1 cells.

Assay sensitivity - cell lysate

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

Lysate was serially diluted in Lysis Buffer and NRAS 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 NRAS expression in less than 200 cells.