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HTRF MAb Anti cmyc-Tb cryptate, 5,000 Assay Points
Tb cryptate-labeled anti-c-Myc antibody for capturing c-Myc-tagged proteins in protein/protein interaction assays.
For research use only. Not for use in diagnostic procedures.
Product Variants
Product information
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.
Specifications
| Assay Points |
5000
|
|---|---|
| Assay Target Type |
Fluorescent reagent
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Therapeutic Area |
Cardiovascular
Infectious Diseases
Inflammation
Metabolism/Diabetes
NASH/Fibrosis
Neuroscience
Oncology & Inflammation
Rare Diseases
|
| Unit Size |
5,000 Assay Points
|
Video gallery
How it works
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-Eu 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, or Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with acceptor 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-Eu 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 details
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 Revvity, is constant and based on the number of tests ordered.
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.
Assay validation
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.
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
Resources
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
SDS, COAs, Manuals and more
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