Large Tumor Suppressor Kinase 1 (LATS1) is a serine/threonine kinase and core component of the Hippo signaling pathway that regulates organ size and tumor suppression. LATS1 is activated by upstream kinases MST1/2 through phosphorylation, leading to autophosphorylation and full activation. Activated LATS1 phosphorylates YAP and TAZ, promoting their cytoplasmic retention and degradation, preventing transcriptional activation of growth-promoting genes. Loss of LATS1 function leads to YAP/TAZ hyperactivation and tumor development in multiple tissues. LATS1 is an important target for understanding tissue growth control and developing therapies for cancers with Hippo pathway dysregulation.
The AlphaLISA SureFire Ultra Human and Mouse Total LATS1 is a sandwich immunoassay for the quantitative detection of total LATS1 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
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.
Induction of LATS1 (Thr1079) phosphorylation
A549 and 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 NIBR-LTSi for 4 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.
NIBR-LTSi treatment resulted in increased LATS1 phosphorylation and decreased levels of Total LATS1.
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.
