HTRF^® Anti-tag Reagents

The HTRF® toolbox reagents include a large selection of anti-tag antibodies for detecting the most commonly used tags. These reagents enable an infinite number of target developments and assay configurations, such as protein:protein and nuclear receptor screening, receptor dimerization, protease assays and investigation of other enzymatic processes. On the right are the assay schemes and/or results of three different assays which are based on HTRF® anti-tag reagents.

HA & c-myc
GABA Receptor Dimerization: γ-aminobutyric acid type B (GABAB) receptor subunits (GB1 and GB2) were 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 then used to construct a receptor activation and dimerization assay. Figure 1 shows how the HA and c-myc tagged subunits are brought into proximity upon receptor dimerization leading to FRET and thus emissions at 665nm. FRET was not seen if a c-myc labeled Vasopressin receptor was used in combination with GABA:HA, showing assay specificity. This assay has been used in subsequent studies by Liu et. al to describe the mechanisms underlying GABAB receptor allosteric modulation.

GST
HDM2/p53 binding assay: The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HDM2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation. Several reports have suggested that disruption of this complex in normal cells results in p53 signaling. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, and two fluorophore-conjugated detection reagents, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody. The HTRF® assay was used to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p73 peptide for HDM2.

DNP
EGFR Kinase Assay: DNP was used to label the poly-GAT kinase substrate. This peptide was incubated with the receptor tyrosine kinase, EGFR. Detection of phosphorylated substrate was performed using XL665-labeled anti-DNP MAb in combination with cryptate-labeled anti-phosphotyrosine (PT66). Figure 3 shows the results when various concentrations of DNP-labeled poly-GAT substrate were used. Further tests demonstrated that Km values were not significantly different when comparing the use of biotinylated versus dinitrophenylated peptide substrates (Km = 66 nM).

Reagents are sold by the number of tests (384-well, 20 µl reactions). Antibodies conjugated to XL665 are supplied on the basis of 20ng of antibody per well. The active moiety per vial is also provided. In practice, the active moiety amount 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.

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