AMPK Methods and Protocols

concentrations to get an initial estimate, and then repeat the

experiment with at least eight concentrations ranging from 0.1

to 10-fold the expectedKm.

41. Traditionally, kinase reactions are initiated with the addition of
    the ATP mixture. However, since AMP is generally used as a
    positive control for AMPK and ATP and AMP can compete for
    binding sites within the protein, we initiate the reaction with
    SAMS peptide to allow nucleotides to achieve equilibrium
    prior to the reaction.


42. Pre-wetting the filter membranes and the simultaneous transfer
    and filtering of samples are essential for data consistency. The
    amount of product retained in the membrane will increase over
    time; therefore, it is important for reactions to be transferred
    simultaneously to the plate.


43. Raw CPM counts can be normalized to product (pM of phos-
    phorylated SAMS) by including a standard curve in each assay.
    A phosphorylated SAMS peptide standard can be produced by
    setting up a reaction at a fixed peptide concentration with a
    single high concentration of AMPK and allowing the kinase
    reaction to go to completion so that nearly 100% of the SAMS
    peptide is phosphorylated. Dilute the mixture and spot onto
    the filter plates to generate the standard curve and convert the
    raw CPM values into concentration units of product.


44. The conditions described above are for determining theKm
    andVmaxfor the SAMS peptide. If the ATP substrate is of
    interest, repeat the experiment above but with varying ATP
    concentrations at saturating SAMS peptide (10
    SAMS pep-
    tideKm).


45. Generate a modified form of full-length human AMPKα 1 β 1 γ 1
    to enable biophysical studies by surface plasmon resonance
    technique. Incorporate an AviTag™ (GLNDIFEAQ-
    KIEWHE) at the N-terminal of theγsubunit and co-express
    with biotin ligase (BirA) in the presence of 50μM biotin (final
    concentration) [9]. Followitems 2– 14 in Subheading2.3 for
    expression and purification of the SPR construct.

Disclosure of Competing Interests:

R.G.K., G.M.W., K.A.B., J.M.W., J.W., A.R.R. and F.R. are employees

of Pfizer Inc., a for-profit global healthcare company. V.D. and P.R.G

declare no competing financial interests.

Biophysical Studies to Evaluate Protein-Ligand Interactions 53