Journal: Journal of biomolecular screening
Article Title: Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay
doi: 10.1177/1087057113478655
Figure Lengend Snippet: A. Magnetic bead pulldown demonstrates interaction of TEM8-mCit with PA. The TEM8-mCit truncation was mixed with PA-biotin and incubated with streptavidin magnetic beads. Significant fluorescence was associated with magnetic beads following removal of solution (blue). A control experiment (red) shows no significant TEM8 fluorescence on beads in the absence of PA. Hence, fluorescence captured on beads reflects TEM8-PA binding. B. Energy transfer is observed upon TEM8-PA binding. TEM8-mCit alone (blue line) displays a donor band at ~525 nm; PA E733C-A546 alone (red) displays an acceptor band at ~572 nm. Upon the addition of PA to TEM8 (green), the donor signal is quenched due fluorescence resonance energy transfer (FRET). A decrease in donor signal intensity at 525 nm is accompanied by an increase in acceptor signal intensity at 572 nm. C. TEM8 affinity for PA is quantified by FRET. TEM8-mCit (donor) signal is quenched upon binding to PA-QSY7 (acceptor). Data were obtained from 96 half-well plates using 500 nm excitation and 535 nm emission filters. Fit of single site binding isotherm to quenching data for this representative data set yielded Kd = 86 nM; the mean of eight independent titrations of TEM8-mCit with PA-QSY7 gave Kd = 108 ± 14 nM (± 95% confidence limit). Unlike ratiometric FRET assays carried out under conditions of constant PA concentration (e.g. the screening assay), affinity measurements require titration of TEM8 with varying concentrations of PA and can be complicated by PA label fluorescence at high PA concentration(s). As a result, affinity measurements here use PA labeled with QSY7, a non-fluorescent FRET acceptor, rather than the fluorescent AF546 used in the screening assay.
Article Snippet: A PA-TEM8-mCit binding assay consisted of mixing PA E733C labeled with biotin-PEG 2 -maleimide (Pierce; labeled according to manufacturer protocols) with TEM8-mCit at 1 μM each in HiHBST.
Techniques: Incubation, Magnetic Beads, Fluorescence, Control, Binding Assay, Förster Resonance Energy Transfer, Concentration Assay, Screening Assay, Titration, Labeling
Vector Laboratories is a verified supplier 
Vector Laboratories manufactures this product 
