Description
Insulinoma-Associated Protein 1 (INSM1) is a zinc finger transcription factor that plays crucial roles in neuroendocrine differentiation and pancreatic islet development. INSM1 regulates expression of genes involved in neurogenesis, hormone production, and cell cycle control. It is transiently expressed during development of neuroendocrine tissues including pancreatic islets and adrenal medulla. INSM1 directly regulates insulin gene expression and is essential for beta cell function. INSM1 serves as a highly sensitive and specific marker for neuroendocrine neoplasms including small cell lung cancer and carcinoid tumors.
The AlphaLISA SureFire Ultra Human Total INSM1 is a sandwich immunoassay for the quantitative detection of total INSM1 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.
Specific expression of INSM1 in NCI-H69 cells
Adherent cells were grown to confluence in a T175 flask at 37°C, 5% CO2, and were lysed with Lysis Buffer at a density of 500,000 cells/mL. Suspension cells were washed and lysed with 5X Lysis Buffer at a density of 200,000 cells/mL.
INSM1 levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 5,000 adherent cells and 2,000 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, Total INSM1 protein was only detected in NCI-H69 lung cancer cells. Negative expression was observed in all cell lines tested in agreement with the literature.
Sensitivity of Total INSM1 assay
Cell lysate was prepared from NCI-H69 cells cultured for four days in a T175 flasks at 37°C, 5% CO2. Cells were harvested, washed with HBSS + 0.1% BSA and lysed at 0.5 x 106 cells/mL with the addition of 5X Lysis Buffer.
Lysate was serially diluted in Lysis Buffer and INSM1 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 per datapoint is indicated. The dotted line represents assay background. This assay can detect INSM1 expression in less than 50 cells/datapoint.
