AMPK Methods and Protocols

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.

1. Dock the streptavidin sensor chip in the Biacore™3000 instru-
   ment and prime the system 3
   with HBS-P buffer.


2. 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.


3. Normalize all four flow cells on the chip surface: Run the
   normalize procedure found under the Tools tab of the Biacore
   software.


4. Switch the buffer system to SPR Buffer B by priming 1
   and
   put instrument in sensorgram mode (found under the Run
   tab).


5. 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.


6. 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.


7. 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 3
   or 2
   dilutions.
   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.


8. 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.

9. 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.