AlphaLISA SureFire Biotin-Free Human Total FOXA1 Detection Kit, 500 Assay Points

FOXA1 recombinant protein dilution

Dilutions of FOXA1 recombinant protein were prepared in Lysis Buffer and FOXA1 levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of diluted 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. Dotted line represents assay background.

Assay specificity - Knockout Validation

FOXA1 levels were assessed in HeLa-WT and FOXA1 KO (Abcam, ab261823) cell lines cultured to confluence in T175 flasks at 37°C, 5% CO₂. Cells were lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was diluted in Lysis Buffer and FOXA1 Total levels were evaluated by AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate (approximately 50,000 cells) 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.

FOXA1 was detected in WT but not KO cells, confirming the specificity of the assay for the detection of FOXA1 protein.

FOXA1 expression in various cell lines

Adherent cells were grown to confluency in a T175 flask in complete medium and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, spun down and lysed with Lysis Buffer at a density of 1.6 x 10⁶ cells/mL.

FOXA1 Total levels were evaluated using the AlphaLISA SureFire Biotin Free 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.

FOXA1 expression was detected in a wide range of human cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HEK293 cells cultured to confluence in a T175 flask and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and FOXA1 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 FOXA1 expression in less than 500 cells.