Description
Overview
Transcription factor EB (TFEB) plays a key role in the regulation of basic cellular processes, including lysosomal biogenesis and autophagy. Dephosphorylated TFEB travels to the nucleus to trigger the transcription of target genes while phosphorylated TFEB exists in the cytoplasm. Mechanistic target of rapamycin (mTOR)-mediated phosphorylation, which takes place at the lysosomal surface, controls the subcellular location and function of TFEB. TFEB is implicated in various diseases, such as lysosomal storage disorders, neurodegenerative diseases, cancers, and metabolic disorders.
The AlphaLISA
SureFire
Human Total TFEB Detection Kit is a sandwich immunoassay for the quantitative detection of total TFEB 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
Alpha SureFire kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
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.
