concentration series of compounds. Total volume for each well
is 200μl. The DMSO standards and the compound samples are
set up from the lowest to highest concentrations. The total
number of compound concentrations is based on whether you
expect to observe binding kinetics (use four compound con-
centrations) or equilibrium binding (use six compound
concentrations).- Add four buffer blanks to the beginning of the plate, followed
by the DMSO standards. - Add two blanks between the DMSO standards and the com-
pound samples, while also including two buffer blanks between
each compound concentration series. - Using the Biacore software, create a method such that all
samples are injected at a flow rate of 50μl/min and 120 s
contact time. DMSO standards and buffer blanks should have a
short dissociation time of 10 s. Compound samples will have
longer dissociation times (generally 300–600 s) based on
anticipated off rates (koff). Compounds need to be fully dis-
sociated before the start of the subsequent injection. - Process the binding responses using Scrubber2 to zero,
x-align, double reference, and correct for excluded volume
effects (DMSO) in the data. - If the association and dissociation phases of compound binding
are observable on the time scale of detection, data is fit to a
simple 1:1 kinetic model using BIAeval to obtain the rate
parameters (kon, koff) and corresponding affinity constant
(KD¼koff/kon) (Fig.5). - If the association and dissociation phases of compound binding
are too fast to measure, KDis determined by fitting the data to
a steady-state affinity algorithm for single site binding, available
within the Scrubber2 software [9, 10, 13].
4 Notes
- Recombinant proteins can be produced by heterologous
expression in several host systems such asE. coli, insect cells,
yeast, or mammalian cells. Biophysical studies require highly
pure and homogenous protein in large amounts. Expression in
E. colior insect cells in general allows one to generate recombi-
nant proteins at reasonable costs while meeting the stringent
purity demands of biophysical studies of AMPK that exists as a
heterotrimer ofα,β, andγsubunits. All of crystallographic and
HDX studies of AMPK that have been reported hitherto
date have utilized protein reagents produced in bacterial or
insect cells.
Biophysical Studies to Evaluate Protein-Ligand Interactions 47