AlphaLISA SureFire Ultra Human and Mouse Total LATS1 Detection Kit, 50,000 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 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.

Effect of Calyculin treatment on LATS1 phosphorylation

PANC-1 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 Calyculin for 2 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LATS1 Phospho (Thr1079) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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.

Calyculin treatment resulted in increased LATS1 phosphorylation and a small decrease in the levels of Total LATS1.

Knockout validation of LATS1 Total assay

LATS1 levels were assessed in THP-1 cells (WT) and LATS1 KO (Abcam, ab277862) cell lines.

Cells were lysed in Lysis Buffer at a density of 2 x 106 cells/mL for 10 minutes at RT with shaking. Lysates were diluted in Lysis Buffer and LATS1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (20,000 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.

LATS1 was detected in WT but not KO cells. This confirms the specificity of the assay for the detection of LATS1 protein.

LATS1 expression in various cell lines

Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 50 µL of lysis buffer. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 1 x 10⁶ cells/mL.

LATS1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (8,000 adherent cells or 10,000 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.

LATS1 expression was detected in a range of human and mouse cell lines.

LATS1 assay sensitivity - cell lysate dilution

Cell lysate was prepared from THP-1 cells treated with 100 nM Calyculin for 3 hours and lysed at 8 x 10⁶ cells/mL.

Lysate was serially diluted in Lysis Buffer and LATS1 Phospho (Thr1079) and Total levels were evaluated by AlphaLISA SureFire Ultra. 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.

Approximate number of cells is indicated. The dotted line represents assay background. This assay can detect LATS1 expression in less than 2,000 cells/datapoint.