Revvity, ALSU-TSKP2-A10K, AlphaLISA Human Total SKP2 Detection Kit, 10,000 Assay Points

Overview
S-phase kinase-associated protein 2 (SKP2) is an F-box protein that functions as a substrate recognition component of the SCF (SKP1–CUL1–F-box) E3 ubiquitin ligase complex. SKP2 targets key cell cycle inhibitors such as p27 Kip1 and p21 Cip1 for ubiquitination and proteasomal degradation, facilitating G1/S transition and cell cycle progression. Overexpression of SKP2 is frequently observed in cancers including breast, prostate, and lung cancer, where it promotes proliferation, metastasis, and resistance to apoptosis. As a driver of oncogenesis, SKP2 is an attractive target for small-molecule inhibitors and protein degradation strategies aimed at restoring cell cycle checkpoints.
The AlphaLISA SureFire Ultra Human Total SKP2 Detection Kit is a sandwich immunoassay for the quantitative detection of total SKP2 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
How it works
Total-AlphaLISA SureFire Ultra assay principle

The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted 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 targeted 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 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 permits the cells 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 AlphaLISA SureFire Ultra quality.
Assay versatility
Versatility of Total SKP2 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 seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 100 µL of Lysis Buffer.
SKP2 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 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.
Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Product Group-Kit
Protocol Time-2h at RT
Sample Volume-10 µL
Shipping Conditions-Shipped in Blue Ice
Target-SKP2
Target Class-Phosphoproteins
Target Species-Human
Technology-Alpha
Therapeutic Area-Oncology
Unit Size-10,000 assay points