Fluorescence anisotropy capillary electrop
horesis (FACE)and affinity probe capillary electrop
horesis (APCE) wit
hlaser-induced fluorescence detection were evaluated foranalysis of peptide-protein interactions wit
h rapid binding kinetics. T
he
Src homology 2 domain of protein SH2-B
![](/images/gifc<font color=)
hars/beta2.gif" BORDER=0 ALIGN="middle"> (SH2-B
![](/images/gifc<font color=)
hars/beta2.gif" BORDER=0 ALIGN="middle"> (525-670)) and a tyrosine-p
hosp
horylatedpeptide corresponding to t
he binding sequence of JAK2were used as a model system. For peptide labeled wit
hfluorescein, t
he
Kd = 82 ± 7 nM as measured byfluorescence anisotropy (FA). APCE assays
had a limit ofdetection (LOD) of 100 nM or 12 amol injected for SH2-B
![](/images/gifc<font color=)
hars/beta2.gif" BORDER=0 ALIGN="middle"> (525-670). T
he separation time of 4 s, ac
hieved usingan electric field of 2860 V/cm on 7-cm-long capillaries,was on t
he same time scale as complex dissociationallowing
Kd (101 ± 12 nM in good agreement wit
h FAmeasurements) and dissociation rate (
koff = 0.95 ± 0.02s
-1 corresponding to a
half-life of 0.73 s) to be determined. T
his measurement represents a 30-fold
hig
herrate of complex dissociation t
han w
hat
had previouslybeen measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, t
he protein was detectedwit
h an LOD of 300 nM or 7.5 fmol injected. FACE wasnot used for determining
Kd or
koff;
however, t
his met
hodprovided better separation resolution for multiple formsof t
he protein t
han APCE. Bot
h met
hods were foundsuitable for analysis of cell lysate. T
hese results demonstrate t
hat FACE and APCE may be useful complementsto existing tec
hniques for exploring binding interactionswit
h rapid kinetics.