Heat Shock Protein 90 (HSP90) is a molecular chaperone essential for the proper folding, stability, and function of numerous client proteins, including many oncoproteins and signaling molecules. HSP90 operates through an ATP-dependent conformational cycle, assisted by co-chaperones, to facilitate protein maturation and prevent aggregation. Client proteins include kinases (AKT, BRAF), transcription factors (p53, HIF-1α), and hormone receptors (estrogen receptor, androgen receptor). HSP90 is constitutively expressed but upregulated under cellular stress conditions. In cancer, HSP90 supports the stability of mutated and overexpressed oncoproteins, making it an attractive therapeutic target. HSP90 inhibitors such as geldanamycin derivatives simultaneously disrupt multiple oncogenic pathways, leading to client protein degradation and tumor cell death, particularly in cancers dependent on HSP90 client proteins.
The AlphaLISA SureFire Ultra Human and Mouse Total HSP90 is a sandwich immunoassay for the quantitative detection of total HSP90 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.
Effect of HSP90 inhibitors in THP-1 cells
THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium and treated with increasing concentrations of SNX-2112 or 17-AAG for 24 hours at 37°C, 5% CO2.
After treatment, the cells were washed with HBSS then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total HSP90 and MCL-1 Phospho (Thr163) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For Total HSP90, lysates were further diluted to work within the assay linear range. For the detection step, 10 µL of cell lysate (approximately 1,000 cells for Total HSP90 or 10,000 cells for Phospho MCL-1) 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, HSP90 inhibitors SNX-2112 and 17-AAG triggered a dose-dependent decrease in the levels of MCL-1 Phospho (Thr163) while Total HSP90 levels remained unchanged.
HSP90 Total assay recognizes alpha and beta isoforms
Reactivity of the Total HSP90 assay for alpha and beta isoforms of the protein was assessed by assaying recombinant proteins.
Dilutions of recombinant HSP90 alpha protein (ab80369) and recombinant HSP90 beta protein (ab80033) were prepared in Dilution Buffer. Total HSP90 was evaluated using AlphaLISA SureFire Ultra. 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 using standard AlphaLISA settings.
The Total HSP90 assay detects both alpha and beta isoforms of the protein.
Versatility of Total HSP90 assay 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.
Lysates were further diluted before evaluation to stay within the assay linear range.
Total HSP90 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (500 adherent cells or 5,000 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.
