HTRF^® Anti-tag Reagents

The HTRF® toolbox reagents include a large selection of anti-tag antibodies labelled with the HTRF tracers Europium or Lumi4®-Terbium Cryptates, and XL665 or d2 for detecting the most commonly used tags. These reagents enable the assay development of an infinite number of target classes and assay formats, such as protein: ligand or protein: protein interactions, nuclear receptor screening, receptor dimerization, kinase and protease assays and investigation of other enzymatic processes. Examples of applications for HTRF® anti-tag reagents are shown in the figures on the right.

HTRF applied to receptor dimerisation at membrane level
GABA Receptor subunits (GB1 and GB2) were co-expressed as two different fusion proteins, GB1 with HA and GB2 with c-myc. Anti-HA and anti-c-myc reagents from the HTRF® toolbox were applied to receptor activation and dimerization assay. Figure 1 shows how the HA and c-myc tags of the GABA_B subunits are brought into proximity upon receptor dimerization thus giving FRET signal. No signal was observed if a c-myc labeled Vasopressin receptor was co-expressed with HA-GABA_B-ASA, showing the assay's specificity. This assay has been used in subsequent studies by Liu et al. to describe the mechanisms underlying GABA_B receptor allosteric modulation.

HTRF anti-GST for protein: protein interaction
The p53, a tumor suppressor protein activated in response to DNA damage, is regulated by the binding of HDM2 which which indeces ubiquitin-medicated degredation of p53. The HTRF® assay was developed to monitor the p53/HDM2 binding, to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p53 homologue, p73, for HDM2. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody.

Lumi4®-Terbium for HTRF GFP and cmyc fused protein assay
Lumi4®-Terbium Cryptate (Tb) conjugates are compatible with either HTRF acceptors such as XL665 or d2 or fluorescein and green fluorescent protein (GFP), thus allowing assay design flexibility to be considerably extended. As an example, HTRF anti-cmyc antibody labeled with Lumi4®-Tb was used for the detection of GFP-and cmyc- fused peptide. As shown in figure 3, a specific signal was only observed with cells expressing GFP and c-myc fused protein. Moreover, using Lumi4®-Terbium Cryptate, the presence of KF in the detection buffer is not mandatory for the measurement. This is an advantage when the ionic strength needs to be maintained at a low level for the interaction to occur. This combination opens new perspectives for the development of HTRF cell-based assays using encoded compatible fluorophores such as GFP.

Reagents are sold by the number of tests (384-well, 20 µl reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracer per vial - see product description sheet). In practice, the amount of active moiety is generally preferred to that of total conjugate as a basis for calculating assay development.

For more information about using HTRF toolbox reagents, click here.