Development of an HTRF method to determine the stoichiometry of subunits...
MAb Anti cmyc-XL665 HTRF®
XL665-labeled anti-c-Myc antibody for capturing c-Myc-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
MAb Anti cmyc-XL665 is an IgG1 raised against a synthetic peptide corresponding to the 408-439 sequence of human c-myc protein labeled with XL. It recognizes the EQKLISEEDL motif and is specific for human c-myc, although a slight cross-reaction has been observed with murine c-myc at high antibody concentration.
This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, 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: MAb Anti cmyc-XL665 binds to the c-Myc 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 c-Myc-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti cmyc-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.
| Motif/Species | Antibody | Active Moiety/5,000 tests | Species and Subtype | Specificity |
|---|---|---|---|---|
| cMYC | MAb 9E10 | 5-10 µg | Mouse IgG1 | EQKLISEEDL peptide |
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.
Applications of HTRF and Tag-lite-lite Assays for HTP Antibody Screening
In collaboration with Bristol Myers Squibb - Scientific Presentations
Academia/Industry Cross-Fertilization through Translational Research
In collaboration with Scripps - Scientific Presentations
HTRF-powered detection of ubiquitin-like protein (UBL) signaling and interactions
In collaboration with Sanford/Burnham - Scientific Presentations
The use of HTRF in biologics discovery
In collaboration with MedImmune - Scientific Presentations
HTRF: One technology, many uses in a pre-clinical laboratory setting
Benefits and considerations of HTRF - Scientific Presentations
Exploiting HTRF for novel drug classes: Stabilizing 14-3-3 protein-protein interactions
In collaboration with Lead Discovery Center GmbH - Scientific Presentations
HTRF a versatil approach for 7TM drug discovery
In collaboration with MRC - 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 cmyc-XL / 61MYCXLF-61MYCXLA-61MYCXLB
61MYCXLF-61MYCXLA-61MYCXLB - 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 cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (DEU) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (ELL) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (ITA) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (SPA) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti cmyc-XL / 61MYCXLA
61MYCXLA - Safety Data Sheet
Safety Data Sheet Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (DEU) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (ELL) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (ITA) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (SPA) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti cmyc-XL / 61MYCXLB
61MYCXLB - Safety Data Sheet
Safety Data Sheet (DEU) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (ELL) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (FRA-FR) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (ITA) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (SPA) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (ENG-GB) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Safety Data Sheet (ENG-US) Anti cmyc-XL / 61MYCXLF
61MYCXLF - Safety Data Sheet
Plate Reader Requirement
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