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AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points

AlphaLISA Surefire Ultra Total Protein
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AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points
AlphaLISA Surefire Ultra Total Protein
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AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
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The AlphaLISA™ SureFire®  Ultra™ Human and Mouse Total IRF5 assay is a sandwich immunoassay for quantitative detection of total IRF5 in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Sample Volume 10 µL
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The AlphaLISA™ SureFire®  Ultra™ Human and Mouse Total IRF5 assay is a sandwich immunoassay for quantitative detection of total IRF5 in cellular lysates using Alpha Technology.

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March lab savings - SAVE 10%. Use promo code below.
MARCH10
Terms and conditions apply.
View product information
Product Variants
Unit Size: 100 assay points
Part #:
ALSU-TIRF5-A-HV
List Price
USD 708.33
Your online price:
Unit Size: 500 assay points
Part #:
ALSU-TIRF5-A500
List Price
USD 2,392.92
Your online price:
Unit Size: 10,000 assay points
Part #:
ALSU-TIRF5-A10K
List Price
USD 14,400.00
Your online price:
Unit Size: 50,000 assay points
Part #:
ALSU-TIRF5-A50K
List Price
USD 46,000.00
Your online price:
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
Total list price:
USD 2,392.92
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AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
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AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points
AlphaLISA Surefire Ultra Total Protein
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AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
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Product information

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

Specifications

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
500 assay points

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AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points
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AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 500 Assay Points
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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.

Assay Principle Total AlphaLISA SureFire Ultra

 

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.

2 plates assay protocol AlphaLISA Surefire Ultra
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.

1 plate assay protocol AlphaLISA Surefire Ultra

Assay validation

Validation of Phospho (Ser446)/Total IRF5 in CpG-B treated cells

RPMI 8226 cells were seeded in a 96-well plate (500,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of CpG-B, ODN-2006 or ODN-1668 (MedChem Express, HY-150218, HY-150726 respectively) for 6 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 cells for ODN-2006 experiment and 20,000 cells for ODN-1668 experiment) 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.

As expected, CpG-B triggered a dose-dependent increase in the levels Phospho (Ser446) while Total IRF5 levels remained unchanged.

RPMI 8226 cells treated with CpG B ODN 2006
RPMI 8226 cells treated with CpG B ODN 1668
Validation of Phospho (Ser446)/Total IRF5 in Calyculin A treated cells

THP-1 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of Calyculin A for 2.5 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

As expected, Calyculin A triggered a dose-dependent increase in the levels IRF5 Phospho (Ser446) while Total IRF5 levels remained unchanged.

THP-1 cells treated with Calyculin A
Specificity of IRF5 Total assay

Total IRF5 protein levels were assessed in A549 wild type (WT) and A549 IRF5 knockout (KO) (Abcam, ab301006) cells. Both cell lines were grown to confluency in a T75 flask in medium at 37°C, 5% CO2. Each flask was lysed in 2 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were then evaluated for Total IRF5 using the AlphaLISA SureFire Ultra 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 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, Total IRF5 protein was detected only in A549 WT cells and no signal was detected in the KO IRF5 A549 cell line, demonstrating assay specificity.

IRF5 Total assay specificity

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.

Evaluation of IRF5 Total expression in PBMCs
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.

Differential expression of IRF5 Total in human and mouse cell lines

Resources

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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments

The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay

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