Revvity, ALSU-TGSPT1-A-HV, AlphaLISA Human and Mouse Total GSPT1 Detection Kit, 100 assay points

Overview
G1 to S phase transition 1 (GSPT1), also known as eRF3a (eukaryotic Release Factor 3a), is a translation termination factor that interacts with eRF1 to regulate mRNA stability and degradation. It plays a crucial role in cell cycle progression, proliferation, and protein synthesis termination. GSPT1 contains a GTPase domain essential for its activity and interacts with various factors, including PABP and components of the mTOR signaling pathway. Dysregulation of GSPT1 is implicated in hematologic malignancies and solid tumors, influencing cell proliferation, apoptosis, and metastasis. Overexpression of GSPT1 can lead to enhanced cell growth and survival, contributing to drug resistance in some cancers. Targeting GSPT1 through protein degradation strategies has emerged as a promising therapeutic approach in cancer treatment.
The AlphaLISA SureFire Ultra Human and Mouse Total GSPT1 Detection Kit is a sandwich immunoassay for the quantitative detection of total GSPT1 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
Expression levels of GSPT1 in a variety of cell types
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 for 10 minutes at RT with shaking (350 rpm). Suspension cells were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
Total GSPT1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (4,000 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.
GSPT1 is highly expressed in most cell lines tested.
Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Product Group-Kit
Protocol Time-2h at RT
Sample Volume-30 µL
Shipping Conditions-Shipped in Blue Ice
Target-GSPT1
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-Alpha
Therapeutic Area-Oncology
Unit Size-100 assay points