Sal-Like Protein 4 (SALL4) is a zinc finger transcription factor that plays crucial roles in embryonic development, stem cell maintenance, and cellular reprogramming. SALL4 regulates pluripotency networks by interacting with core transcription factors including OCT4, SOX2, and NANOG, while also modulating chromatin structure through recruitment of epigenetic regulators. It is essential for early embryogenesis and hematopoietic stem cell function. SALL4 expression is normally restricted to embryonic tissues but becomes aberrantly reactivated in various cancers, including acute myeloid leukemia, hepatocellular carcinoma, and germ cell tumors. In malignancies, SALL4 promotes stemness, drug resistance, and metastasis while inhibiting differentiation and apoptosis. Its oncofetal expression pattern makes SALL4 an attractive target for cancer therapy and a valuable biomarker for diagnosis and prognosis.
The AlphaLISA SureFire Ultra Human Total SALL4 is a sandwich immunoassay for the quantitative detection of total SALL4 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.
SALL4 degradation using molecular glue compounds
BeWo 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 NVP-DKY709 for 24 hours.
After treatment, the cells were washed in HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) and further diluted before loading. Total SALL4 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells for Total SALL4 and 200 cells for Total Cofilin) 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.
In agreement with literature, NVP-DKY709 degraded around 50% of SALL4 protein while Total Cofilin levels remained unchanged.
BeWo cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Thalidomide for 24 hours.
After treatment, the cells were washed in HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) and further diluted before loading. Total SALL4 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,000 cells for SALL4 and 100 cells for Cofilin) 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.
As expected, Thalidomide treatment resulted in a dose-dependent decrease in the levels of Total SALL4.
SALL4 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 100 µL of Lysis Buffer. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 4 x 106 cells/mL.
SALL4 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension 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.
As expected, SALL4 expression was low or undetectable in most of the cell lines tested. Moderate expression levels were observed in BeWo cells.
