AlphaLISA SureFire Biotin-Free Human and Mouse Total SMARCA2 Detection Kit, 500 Assay Points

Specificity of Total SMARCA2 assay

Total SMARCA2 expression levels were assessed in parallel with Total SMARCA4 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.

SMARCA2 expression in various cell lines

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

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SMARCA2 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.

SMARCA2 is expressed in a wide variety of cell lines. Very high levels of expression were detected in HeLa, U937 and THP-1 cells.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HeLa cells seeded in a T175 flask and cultured to confluence and lysed in 5 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and SMARCA2 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 SMARCA2 expression in less than 250 cells/datapoint.