AlphaLISA SureFire Biotin-Free Human and Mouse Total SMARCA4 Detection Kit, 500 Assay Points

Specificity of Total SMARCA4 assay

Total SMARCA4 expression levels were assessed in parallel with Total SMARCA2 in a panel of relevant cell lines.

SMARCA2 and SMARCA4 Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells and 40,000 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.

As expected, SMARCA2 and SMARCA4 expression is dependent upon cell type. SMARCA2 is expressed in A549 cells, whilst SMARCA4 is undetectable. Conversely, SMARCA4 is expressed in Caco-2 cells where SMARCA2 is undetectable.

These results demonstrate the specificity of the SMARCA2 and SMARCA4 assays as these 2 proteins share more than 70% sequence identity.

SMARCA4 expression in various cell lines

Adherent cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO_2. Suspension cells were harvested, washed, and seeded in a 96-well plate (400,000 cells/well).

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SMARCA4 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells and 40,000 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.

SMARCA4 is highly expressed in a wide variety of cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from SH-SY5Y cells cultured to confluence in a T175 flask 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 using the AlphaLISA SureFire Biotin Free 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 SMARCA4 expression in less than 300 cells/datapoint.