Description
Streptavidin has been labeled with XL665. The streptadivin-XLent! (SA-XLent!) results from a special in-house conjugation process leading to a high grade conjugate with enhanced fluorescent properties. Biotin binds to Streptavidin with high affinity (Ka=1015M-1). The binding is rapid and stable, making it an ideal choice for use in a variety of assays such as enzyme assays, protein-protein binding assays and molecular biology assays.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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: Streptavidin-XLent! binds to the biotinylated partner A while partner B* binds to a specific antibody labeled to an HTRF donor. *partner B can also be tagged, Fc fused. In these cases use for the detection, the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin), labeled with donor.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a biotinylated-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add Streptavidin-XLent! (5 µL) and anti-partner B labeled with acceptor (5 µL), incubate and read. *partner B can also be tagged, Fc fused or directly labeled. In these cases use for the detection, the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin), labeled with donor.
HTRF Tyrosine kinase assay
HTRF reagents (Europium-Cryptate PT66 and Streptavidin-XLent!) and conjugates from two other suppliers were used in an HTRF tyrosine kinase assay. When compared to competitor, Streptavidin-XLent! shows overall superior performance.
SaXLent! compared to SaXL on the EGFR tyrosine kinase assay
Increasing quantities of EGFR were incubated with biotinylated polyGAT (50 nM). The enzymatic reaction was stopped using EDTA following a 20 minute incubation. Europium-labeled PT66 (20 ng/well) and Streptavidin-XL665 (50 nM) or StreptavidinXLent! (50 nM) were added. HTRF signal was detected following a 20 minute incubation. In addition to the increased sensitivity, over 25% less enzyme was required when using streptavidin-XLent! to produce identical assay signals.
