Description
Protein Kinase C theta (PKCθ) is a calcium-independent, diacylglycerol-activated serine/threonine kinase that plays a central role in T cell activation and immune responses. PKCθ is recruited to the immunological synapse upon T cell receptor (TCR) engagement, where it phosphorylates key substrates including CARMA1, leading to NF-κB activation and IL-2 production. It is essential for T cell proliferation, differentiation, and effector function, particularly in CD4+ T helper cells and CD8+ T cells. PKCθ also regulates T cell survival and memory formation. Dysregulation of PKCθ contributes to autoimmune diseases, transplant rejection, and T cell malignancies. Selective PKCθ inhibitors are being developed as immunosuppressive agents for autoimmune disorders and as potential cancer therapeutics to modulate T cell-mediated immunity and overcome immune evasion mechanisms.
The AlphaLISA SureFire Ultra Human and Mouse Total PKCθ is a sandwich immunoassay for the quantitative detection of total PKCθ 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.
Expression of PKCθ 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 200 µL of Lysis Buffer.
Suspension cells were seeded at 800,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 200 µL of Lysis Buffer. PKCθ levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 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, high PKCθ levels were observed in Jurkat, HEL 92.1.7 and EL-4 cells. No signal was detected in Raji and HeLa cells.
Selectivity of PKCθ Total assay
Selectivity of the Total PKCθ assay was assessed by assaying recombinant PKCθ protein.
Dilutions of recombinant PKCθ protein (Abcam ab56641) were prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay. 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.
