AlphaLISA SureFire Ultra Human and Mouse Total SMARCA-4 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 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.

HeLa 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 increasing concentrations of ACBI1 for 4 hours with or without pretreatment with 2 µM MG132 for 30 minutes.

After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SMARCA4 levels were evaluated by AlphaLISA SureFire Ultra. 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, ACBI1 treatment resulted in a dose-dependent decrease in the levels of SMARCA4. The lack of protein degradation in the presence of MG132 confirmed that the degradation was driven by the ubiquitin-proteosome system.

Knockout validation of SMARCA4 Total assay

SMARCA4 levels were assessed in WT HEK293T cells and SMARCA4 KO (Abcam, ab255432) cells seeded at decreasing cell density in a 96-well plate in complete medium, and incubated overnight at 37°C, 5% CO₂.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SMARCA4 levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximate number of cells is indicated) 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, SMARCA4 was only detected in WT cells confirming the specificity of the assay.

Differential expression of SMARCA4 and SMARCA2 in relevant cell lines

SMARCA2 and SMARCA4 expression levels were assessed in a panel of relevant cell lines 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.

As expected, SMARCA2 and SMARCA4 expression is dependent upon cell type. SMARCA2 is expressed in THP-1 cells, whilst SMARCA4 is undetectable. Conversely, SMARCA4 is expressed in Caco-2 cells where SMARCA2 is undetectable. These results demonstrate the specificity of the SMARCA4 assay despite sharing more than 70% sequence identity with SMARCA2.

Sensitivity of the SMARCA4 assay was assessed by evaluating SMARCA4 recombinant protein. Serial dilutions of recombinant SMARCA4 protein were prepared in Lysis Buffer and assayed 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.

Cell lysate was prepared from SH-SY5Y cells cultured to confluence in T175 flasks and lysed in 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and SMARCA4 levels were evaluated 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/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect SMARCA4 expression in less than 500 cells/datapoint.