Description
AT-Rich Interaction Domain 1B (ARID1B) is a chromatin remodeling protein and core subunit of the SWI/SNF complex that regulates gene expression through ATP-dependent nucleosome repositioning. ARID1B is particularly important for neural development, where it regulates neuronal differentiation, synaptic function, and brain morphogenesis. Haploinsufficiency of ARID1B causes Coffin-Siris syndrome, characterized by intellectual disability and developmental delay. ARID1B mutations are also found in various cancers including neuroblastoma and breast cancer, where loss of function contributes to tumor progression. The tumor suppressor function of ARID1B makes it an important target for developing epigenetic therapies.
The AlphaLISA SureFire Ultra Human and Mouse Total ARID1B is a sandwich immunoassay for the quantitative detection of total ARID1B 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.
ARID1B expression in various cell lines
Expression of ARID1B was evaluated in various cell lines using the AlphaLISA SureFire Ultra assay kit.
For the detection step, 10 µL of lysate (approximately 5,000 adherent cells and 8,000 suspension 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.
ARID1B was expressed at high levels in all cell lines tested. Similar levels of ARID1B protein were detected in HEK293T (ARID1A-KO) and HEK293T wild type cell lines.
Total ARID1B assay sensitivity - cell lysate dilution
Cell lysate was prepared from HCT-116 cells cultured to confluence in a T175 flasks and lysed in 16 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and ARID1B 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 ARID1B expression in less than 100 cells/datapoint.
