Revvity, ALSU-TIRF5-A10K, AlphaLISA SureFire Ultra Human and Mouse Total IRF5 Detection Kit, 10,000 Assay Points

Overview
Interferon regulator factor 5, IRF5, is a transcriptional regulator of type I interferon (IFN-alpha and IFN-beta)-dependent immune responses. This transcription factor plays a key role in the innate immune response against DNA and RNA viruses. IRF5 is present in the cytoplasm of uninfected cells in an inactive form. Various mediators of the pathways that follow viral infection can overexpress and phosphorylate IRF5. IRF5 is associated with various cancers and autoimmune diseases.
The AlphaLISA SureFire Ultra Human and Mouse Total IRF5 Detection Kit is a sandwich immunoassay for the quantitative detection of total IRF5 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.
Assay versatility
Evaluation of IRF5 Total expression in Peripheral Blood Mononuclear Cells
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll® Plaque Plus (Merck GE17-1440-02).
Cells were seeded in 96-well plate (400,000 cells/well) and lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Generated lysate was then serially diluted in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra Total IRF5. For the detection step, 10 µL of cell lysate (starting from approximately 40,000 cells) were 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. The number of cells/datapoint is indicated on graph.

Differential expression of IRF5 Total in various cell lines
Human and mouse cell lysates were diluted with Lysis Buffer. Approximate number of cells/datapoint is indicated on graph. Total IRF5 levels were evaluated using the AlphaLISA SureFira Ultra assay kit. For the detection step, 10 µL of cell lysate 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.
Total IRF5 expression varies depending upon cell type. As expected, a very high level of expression was detected in human cell lines: THP-1, KARPAS-299 and U937 and mouse cell lines: RAW 264.7 and L929.
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-IRF5
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-Alpha
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points