Tb cryptate-labeled anti-c-Myc antibody for capturing c-Myc-tagged proteins in protein/protein interaction assays.
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Tb cryptate-labeled anti-c-Myc antibody for capturing c-Myc-tagged proteins in protein/protein interaction assays.
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Overview

MAb Anti cmyc-Tb is an IgG1 raised against a synthetic peptide corresponding to the 408-439 sequence of human c-myc protein labeled with Tb. 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-Tb cryptate binds to the c-Myc tagged partner A while partner B* binds to a specific Ab labeled with an HTRF acceptor. *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 acceptor for the detection.
assay principle of detection assay using HTRF MAb Anti cmyc-Tb cryptate

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-Tb cryptate (5 µL) and anti-partner B labeled with acceptor (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 acceptor for the detection.
assay protocol of detection assay using HTRF MAb Anti cmyc-Tb cryptate

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.
description of the active moiety in an HTRF conjugate

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.

GFP-c-Myc fused protein assay - assay principle

Terbium Cryptate (Tb) conjugates are compatible with either HTRF acceptors such as XL665 or d2, or fluorescein and green fluorescent protein (GFP), meaning assay design flexibility can be considerably extended. As an example, HTRF anti-cmyc antibody labeled with -Tb was used for the detection of GFP-and cmyc- fused peptide. A specific signal was only observed with cells expressing GFP and c-myc fused protein.This combination opens new perspectives for the development of HTRF cell-based assays using encoded compatible fluorophores such as GFP.
assay principle and results of the HTRF GFP/c-Myc assay

GFP-c-Myc fused protein assay - results

HTRF GFP- and cmyc fused protein assay. Living cells were dispensed into a 384-well plate and transiently transfected with plasmid encoding for GFP-and cmyc fused peptide. After a 24h incubation at 37ºC, anti-cmyc-Tb in lysis buffer was added, and the signal was read on PHERAstar after a further hour of incubation at RT. A signal to background higher than 3 was observed when comparing cells transfected with GFP-c-myc plasmid to the empty vector
results of the HTRF GFP/c-Myc assay
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

Product Insert Anti cmyc-Tb / 61MYCTAF-61MYCTAA-61MYCTAB

61MYCTAF-61MYCTAA-61MYCTAB - 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

Cisbio Product Catalog 2019

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

Safety Data Sheet Anti cmyc-Tb / 61MYCTAA

61MYCTAA - Safety Data Sheet

Safety Data Sheet Anti cmyc-Tb / 61MYCTAB

61MYCTAB - Safety Data Sheet

Safety Data Sheet Anti cmyc-Tb / 61MYCTAF

61MYCTAF - Safety Data Sheet

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