Description
The kit enables the rapid detection of Flag-tagged fusion proteins at every stage of protein production and purification. It can be used to verify that the Flag motif of a fusion protein is accessible to the anti-FLAG antibody or to estimate the concentration of a Flag-tagged protein. When calibrated using the appropriate protein, the assay is remarkably accurate and compatible with the SOPs. It can also be used to eliminate false positives.
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
As shown in the graph, Flag peptide labeled with d2 is detected by the anti Flag-Eu Cryptate conjugate. The Flag-tagged fusion protein to be ascertained competes with the Flag-peptide-d2 conjugate for binding to the anti Flag-Eu Cryptate conjugate. The specific signal is inversely proportional to the concentration of Flag-tagged fusion protein.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume. Dispense 10 µL of standard or of Flag-tagged protein of interest into a well, with 5 µL of anti Flag-Eu Cryptate conjugate and 5 µL of Flag peptide-d2 conjugate. Incubate for 1 hour at RT, and then read.
FLAG check kit performance
The Flag Check Kit enables the rapid detection of FLAG-tagged fusion proteins. It can be used either to ensure that the Flag motif of a fusion protein is accessible to the anti-FLAG antibody, or to approximate the concentration of a FLAG-tagged protein.
FLAG check kit performance on various tag positions
The Flag Check kit enables the detection of Flag tagged proteins, whatever the tag insertion (N-terminal, C-terminal, or internal Flag tags).
