Revvity, ASBF-TCYCE1-A50K, AlphaLISA Biotin-Free Human Total Cyclin E1 Detection Kit, 50,000 Assay Points

Overview
Cyclin E1 is a regulatory protein that controls the G1-to-S phase transition of the cell cycle by activating cyclin-dependent kinase 2 (CDK2). The Cyclin E1-CDK2 complex phosphorylates substrates including retinoblastoma protein and p27Kip1, promoting initiation of DNA replication. Cyclin E1 overexpression or amplification occurs in numerous cancers including breast, ovarian, and endometrial carcinomas, driving uncontrolled proliferation and chromosomal instability. Low molecular weight forms of Cyclin E1 exhibit enhanced oncogenic activity and resistance to CDK inhibitors. Selective CDK2 inhibitors are being developed as therapeutics for Cyclin E1-amplified cancers.
The AlphaLISA SureFire Biotin-Free Human Total Cyclin E1 Detection Kit is a sandwich immunoassay for the quantitative detection of total Cyclin E1 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 Biotin Free kits are compatible with:

Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples

AlphaLISA SureFire Biotin Free kits can be used for:

Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
How it works
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA Surefire Biotin Free assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA Surefire 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. 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 Biotin Free 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 Biotin Free 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 one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Biotin Free 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 quality.
Assay sensitivity
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from BeWo cells cultured to confluency in T175 flasks at 37°C, 5% CO2. Each flask was lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and Cyclin E1 Phospho (Thr77) and Total levels were evaluated by AlphaLISA SureFire Biotin Free. For the detection step, 10 µL of 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 using standard AlphaLISA settings.
Approximate number of cells per datapoint is indicated. The dotted line represents assay background. This assay can detect Cyclin E1 Total down to 100 cells/datapoint.
Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Protocol Time-2h at RT
Sample Volume-10 µL
Shipping Conditions-Shipped in Blue Ice
Target-Cyclin E1
Target Class-Phosphoproteins
Target Species-Human
Technology-Alpha
Therapeutic Area-Oncology
Unit Size-50,000 Assay Points