SMARCA2 (Probable global transcription activator SNF2L2), a member of the SWI/SNF family, plays a crucial role in chromatin remodeling and DNA damage repair. It serves as an ATPase subunit within the SWI/SNF complex, which regulates genes involved in DNA replication and cell proliferation. Notably, SMARCA2 and SMARCA4 are well-established in SMARCA4-deficient cancers, where SMARCA2 inactivation leads to tumor cell death. Considering its potential, SMARCA2 could be targeted for protein degradation therapeutics in cancer treatment.
The AlphaLISA SureFire Ultra Human Total SMARCA2 Detection Kit is a sandwich immunoassay for the quantitative detection of total SMARCA2 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
Alpha SureFire kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput drug screening
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.
PROTAC induced SMARCA2 degradation
HeLa 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 ACBI1 at the indicated concentrations for 4 hours.
After treatment, the cells were lysed with 100µL of Lysis Buffer for 10 minutes at RT with shaking at 350 rpm. SMARCA2 and SMARCA4 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate(approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5µL 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 Nexus™ using standard AlphaLISA settings.
As expected, ACBI1 induced a dose-dependent decrease in both SMARCA2 and SMARCA4 levels.
Selectivity of SMARCA2 Total assay
Total SMARCA2 protein levels were assessed in HeLa cells (WT) and SMARCA2 knockout (KO) HeLa cells. SMARCA2 KO cells (Abcam ab265416) and WT-HeLa cells were seeded in a 96-well plate in complete medium, and incubated overnight at 37°C, 5% CO2. After treatment, the cells were lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). SMARCA2 Total levels were then 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 Nexus using standard AlphaLISA settings.As expected, SMARCA2 was detected in the WT-Hela cells but not in the SMARCA2-KO cell line.
Total SMARCA2 expression levels were assessed in parallel with Total SMARCA4 in a panel of relevant cell lines. Lyophilized cell lysates from various cell lines were resuspended and then diluted in Lysis Buffer to ensure that detection was within the dynamic range of the kits (data not shown). SMARCA2 and SMARCA4 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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 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 Nexus using standard AlphaLISA settings.
As expected, SMARCA2 and SMARCA4 expression is dependent upon cell type. SMARCA2 is expressed in THP1 and A549 cells, whilst SMARCA4 is undetectable. Conversely, SMARCA4 is expressed in Caco2 cells where SMARCA2 is undetectable. These results demonstrate the selectivity of the SMARCA2 assay over SMARCA4, despite these 2 proteins sharing more than 70% sequence identity.
Selectivity of the Total SMARCA2 assay for SMARCA4 protein was assessed by assaying recombinant SMARCA4 protein. Dilutions of recombinant SMARCA4 protein (ab82237) were prepared in Lysis Buffer. SMARCA2 and SMARCA4 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of protein 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 Nexus using standard AlphaLISA settings.
As expected, the Total SMARCA2 signal was equivalent to the non-specific signal (dotted line). These results demonstrate the selectivity of the SMARCA2 assay over SMARCA4, despite these 2 proteins sharing more than 70% sequence identity.
