Revvity, ALSU-TBTK-A500, AlphaLISA Human and Mouse Total BTK Detection Kit, 500 Assay Points

Overview
Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that plays a critical role in B-cell receptor (BCR) signaling, which is essential for B-cell development, differentiation, and survival. BTK is involved in the activation of downstream signaling pathways including NF-κB, MAPK, and PLCγ2, and is a key regulator of immune responses. Dysregulation or mutation of BTK is associated with immunodeficiencies such as X-linked agammaglobulinemia (XLA) and various B-cell malignancies. BTK has emerged as a prominent therapeutic target, with several BTK inhibitors approved or in development for the treatment of hematologic cancers and autoimmune diseases.
The AlphaLISA SureFire Ultra Human and Mouse Total BTK Detection Kit is a sandwich immunoassay for the quantitative detection of total BTK 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.

In the AlphaLISA™ SureFire® Ultra™ assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of protein present in the sample.
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.
Assay versatility
Versatility of Total BTK 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 200 µL of Lysis Buffer.
Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate and lysed with 200 µL of Lysis Buffer.
BTK levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 cells (adherent) or 20,000 cells (suspension)) 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, BTK expression is measured only in myeloid and lymphoid cells.
Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Lysis Buffer Compatibility-Lysis Buffer
Molecular Modification-Total
Product Group-Kit
Sample Volume-10 µL
Shipping Conditions-Shipped in Blue Ice
Target-BTK
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-Alpha
Unit Size-500 assay points