3.5 Surface Plasmon
Resonance (SPR)
Design a suitable SPR construct for the immobilization of AMPK on
streptavidin chips(seeNote 45).
Followsteps 1– 6 below for capturing AMPK onto streptavidin-based
SPR chips.
- Dock the streptavidin sensor chip in the Biacore™3000 instru-
ment and prime the system 3with HBS-P buffer. - Condition all four flow cells on the chip surface. Set the flow
rate to 100μl/min and inject 50μl of conditioning solution
three consecutive times using the Quickinject with Extraclean
option for the injections. - Normalize all four flow cells on the chip surface: Run the
normalize procedure found under the Tools tab of the Biacore
software. - Switch the buffer system to SPR Buffer B by priming 1and
put instrument in sensorgram mode (found under the Run
tab). - Dilute the protein to 1.25μg/ml in Buffer B and inject onto
flow cells 2 and 3 at a rate of 5μl/min in order to achieve
~4000–6000 RU of binding to the chip surface. - Flow Buffer B over all four flow cells of the chip surface to
equilibrate the chip surface at a flow rate of 5μl/min for
approximately 4 h before collecting compound binding data. - Prepare compound solutions with starting compound concen-
trations of at least 20-fold over the known EC 50 value while still
maintaining 2% DMSO in the solutions. The final compound
solution is then diluted serially in either 3or 2dilutions.
Use the equationC 1 V 1 ¼C 2 V 2. whereC 1 is the concentration
of compound stock in DMSO,V 1 volume of DMSO,C 2 start-
ing concentration of compound for serial dilution, andV 2 final
volume of compound solution. Solve forC 1 to determine the
compound stock concentration in DMSO. Example: Com-
pound X is made up in DMSO stock solutions of 50μM,
16.7μM, 5.55μM, and 1.85μM. Add 4μl of each compound
concentration to 196μl of Buffer A, resulting in a final DMSO
concentration of 2% (v/v) and final compound concentrations
of 1μM, 333 nM, 111 nM, and 37 nM. - Prepare a DMSO sample curve by adding 5, 10, 20, 30, and
40 μl of DMSO to 1 ml of assay Buffer A, resulting in final
concentrations of 0.5, 1, 2, 3, and 4% DMSO.
Followsteps 9– 15 below for acquisition of SPR data of compounds
and their analysis.
- Assemble a 96-well sample Greiner Bio-One Plate to include
the following samples: the DMSO standard curve samples,
buffer blanks consisting of Buffer B alone, and each
46 Ravi G. Kurumbail et al.