HTRF used for the foundation of a cellular Ser/Thr-kinase platform that ...
MAb Anti cmyc-Eu cryptate HTRF®
- High affinity
MAb Anti cmyc-Eu is an IgG1 raised against a synthetic peptide corresponding to the 408-439 sequence of human c-myc protein labeled with Eu. 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 concentrations.
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.
- DETECTION OF LARGE COMPLEXES
- BATCH-TO-BATCH REPRODUCIBILITY
- LOW TO HIGH KD DETECTION
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.
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.
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.
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.
In collaboration with Bristol Myers Squibb - Scientific Presentations
In collaboration with Scripps - Scientific Presentations
In collaboration with Sanford/Burnham - Scientific Presentations
Benefits and considerations of HTRF - Scientific Presentations
In collaboration with Lead Discovery Center GmbH - Scientific Presentations
In collaboration with MRC - Scientific Presentations
Benefit from unlimited flexibility for your assay development - Flyers
In collaboration with Boehringer Ingelheim - Posters
Every interaction is within range - Technical Notes
Easy pharmacological characterization of PPI modulators. - Technical Notes
Deciphering low- and high affinity interactions - Application Notes
Monitoring nuclear receptor binding with HTRF assays - Application Notes
Challenge large complexes with HTRF assays - Application Notes
Get the brochure about technology comparison. - Brochures
See how peer researchers challenge the viral life cycle with PPI assays - Application Notes
Plate Reader Requirement
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