HTRF, a generic mthod in the study of protein-protein interactions
PAb Anti Human IgG-XL665 HTRF®
XL665-labeled anti-human IgG antibody for capturing human IgG-tagged proteins in protein/protein interaction assays.
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No-wash
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Ease-of-use
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High affinity
Overview
IgGs from goat anti-Human immunoserum were immunopurified and labeled with XL665. PAb Anti Human IgG-XL665 may be used to detect the binding of unlabeled human specific antibody on a target molecule or on Ig fusion proteins.
This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.
HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.
Benefits
- LARGE COMPLEXES DETECTION
- BATCH-TO-BATCH REPRODUCIBILITY
- LOW TO HIGH KD DETECTION
Assay principle
In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: PAb Anti Human IgG-XL665 binds to the Human Fc fused tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with donor for the detection.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a Human Fc fused-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add PAb Anti Human IgG-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with donor for the detection.
Build HTRF interaction assays for your specific application
Reagents are sold by the number of tests (20 µL reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20 ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracers per vial - see product description sheet). The active moiety is defined as the active part of a conjugate (e.g. antibody).
How do the number of tests relate to active moiety?
The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Cisbio, is constant and based on the number of tests ordered.
Species Antibody Species Specificity Active Moiety 5,000 tests Europium conjugates XL665 conjugates Human immunoglobulins PAb Goat Human Fc Igs 10 µg 100 µg
Recommended quantities of Cryptate and XL665 conjugates
Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.
CD28/CD86 binding assay
CD28 provides a major costimulatory signal for CD4-positive T cells. Ligation with its natural ligands CD80 (B7-1) and CD86 (B7-2) leads to signals during activation that are required for the production of interleukin-2. HTRF reagents were used to develop an assay for antagonists of CD28 and CD86 binding. Both molecules were respectively tagged with human and rat IgG Fc fragments, and detected using the XL665-labled anti-human antibody. CD86 was expressed as a fusion protein with a rat Ig domain which is recognized by a biotinylated antibody, followed by binding of streptavidin-Eu cryptate.
Mellor et al. Development of a CD28/CD86 (B7-2) binding assay for high throughput screening by homogenous time-resolved fluorescence. J Biomol Screening. 1998;3: 91-99.
Applications of HTRF and Tag-lite-lite Assays for HTP Antibody Screening
In collaboration with Bristol Myers Squibb - Scientific Presentations
The use of HTRF in biologics discovery
In collaboration with MedImmune - Scientific Presentations
HTRF protein-protein interaction reagents
Benefit from unlimited flexibility for your assay development - Flyers
Addressing the interactome with protein-protein assays
Cover all PPIs with one approach - Brochures
HTRF -A Beneficial Tool for Lead Optimization of Biotherapeutics
In collaboration with Boehringer Ingelheim - Posters
The ultimate guide to PPI assays with HTRF
Complete guide - Guides
Guidelines for optimizing protein-protein interaction assays using HTRF PPI reagents
Every interaction is within range - Technical Notes
Product Insert Anti (h) IgG-XL / 61HFCXLF-61HFCXLA-61HFCXLB
61HFCXLF-61HFCXLA-61HFCXLB - Product Insert
HTRF PPI your dream assay served on a plate
Sandwiches aren't just for eating - Infographics
Best practices for pharmacological characterization of PPI inhibitors
Easy pharmacological characterization of PPI modulators. - Technical Notes
HTRF assays handle low- to high affinity protein-protein interactions
Deciphering low- and high affinity interactions - Application Notes
Nuclear receptor ligand identification with HTRF
Monitoring nuclear receptor binding with HTRF assays - Application Notes
HTRF addresses large protein-protein interaction complexes
Challenge large complexes with HTRF assays - Application Notes
A brief history of Protein-Protein Interactions
How well do you know PPI? - Infographics
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Virology research solutions using HTRF Protein-Protein Interaction assays
See how peer researchers challenge the viral life cycle with PPI assays - Application Notes
Advance your CAR-T cell research - Flyers
Safety Data Sheet Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (DEU) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (ELL) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (ITA) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (SPA) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti (h) IgG-XL / 61HFCXLA
61HFCXLA - Safety Data Sheet
Batch Information Anti (h) IgG-XL / 20220715
61HFCXLA Batch 13D - Batch Information
Batch Information Anti (h) IgG-XL / 20240715
61HFCXLA Batch 13D - Batch Information
Batch Information Anti (h) IgG-XL / 20250209
61HFCXLA Batch 13E - Batch Information
Batch Information Anti (h) IgG-XL / 20230519
61HFCXLA Batch 13E - Batch Information
Safety Data Sheet Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (ELL) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (ITA) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (SPA) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Safety Data Sheet (DEU) Anti (h) IgG-XL / 61HFCXLB
61HFCXLB - Safety Data Sheet
Batch Information Anti (h) IgG-XL / 20230519
61HFCXLB Batch 13A - Batch Information
Safety Data Sheet (DEU) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (ELL) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (ITA) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (SPA) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti (h) IgG-XL / 61HFCXLF
61HFCXLF - Safety Data Sheet
Batch Information Anti (h) IgG-XL / 20230519
61HFCXLF Batch 13C - Batch Information
Plate Reader Requirement
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