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AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 Assay Points

AlphaLISA Surefire Ultra Total Protein
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AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 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 Total PAX5 assay is a sandwich immunoassay for quantitative detection of total PAX5 in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Ultra™ Human Total PAX5 assay is a sandwich immunoassay for quantitative detection of total PAX5 in cellular lysates using Alpha Technology.

View product information
Product Variants
Unit Size: 100 assay points
Part #:
ALSU-TPAX5-A-HV
Unit Size: 500 assay points
Part #:
ALSU-TPAX5-A500
Unit Size: 10,000 assay points
Part #:
ALSU-TPAX5-A10K
Unit Size: 50,000 assay points
Part #:
ALSU-TPAX5-A50K
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).
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AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
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AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
Video Play Button
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
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Product information

Overview

PAX5 is a transcription factor crucial for B-cell development, regulating genes specific to B-cell identity. It interacts with transcription factors like EBF1 and RUNX1 to control gene expression during B-cell differentiation. Alterations in PAX5, including mutations and translocations, are implicated in B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma, disrupting normal B-cell function and driving malignancy.

The AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit is a sandwich immunoassay for the quantitative detection of total PAX5 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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
PAX5
Target Class
Phosphoproteins
Target Species
Human
Technology
Alpha
Therapeutic Area
Oncology
Unit Size
50,000 assay points

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AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 Assay Points
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AlphaLISA SureFire Ultra Human Total PAX5 Detection Kit, 50,000 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.

2plates-assay-protocol-AlphaLISA-Surefire-Ultra.jpg
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

Specificity of PAX5 Total assay

Specificity of the Total PAX5 assay was assessed by assaying recombinant PAX2 and PAX5 proteins. Dilutions of recombinant PAX2 and PAX5 proteins were prepared in Lysis Buffer. PAX5 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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 PAX5 assay was specific only to PAX5 recombinant protein, no signal was detected with PAX2 recombinant protein. This result demonstrates the specificity of the Total PAX5 SureFire Ultra assay as these two proteins share approximately 70% sequence identity.

Selectivity of PAX5 Total Assay
Validation of PAX5 Total 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 100 µL of Lysis Buffer.
Suspension cells (Daudi, RAMOS) were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 100 µL of Lysis Buffer. Suspension cell lysates were further diluted 1:2.5 with Lysis Buffer.

PAX5 Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 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.

Total PAX5 expression is dependent upon cell type. As expected, a very high level of expression was detected in Daudi and Ramos cells, while signal was undetectable in OVCAR-3, AN3 CA, PANC-1 cells.

Validation of PAX5 Total Assay in Various Cell Lines

Resources

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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments

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Species compatibility for HTRF, AlphaLISA SureFire Ultra and Alpha SureFire Ultra Multiplex assays

This document includes detailed tables listing HTRF™, AlphaLISA™ SureFire® Ultra™, and Alpha SureFire® Ultra™ Multiplex assays...

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