Description
MAb Anti HA-XL665 is an IgG2b raised against the twelve-amino acid peptide sequence CYPYDVPDYASL labeled with XL665. It recognizes the influenza virus hemagglutinin epitope (YPYDVPDYA) which has been used extensively as a general epitope tag in expression vectors.
This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, receptor/ligand.
HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.
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: MAb Anti HA-XL665 binds to the HA tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, 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 HA-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti HA-XL665 (5 µL) and anti-partner B labeled with donor (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 donor for the detection.
