AMPK Methods and Protocols

8. 1phosphate buffer saline (PBS): 138 mM NaCl, 2.7 mM
   KCl, 1.47 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 ,pH7.


9. Cell scraper.


10. Lysis buffer: 50 mM HEPES, pH 7.5, 50 mM KF, 1 mM KPi,
    5 mM EDTA, 5 mM EGTA, 5 mMβ-mercaptoethanol, 1 mM
    vanadate, standard protease inhibitor mixture (complete mini,
    Roche), 1% (v/v) Triton X-100 (seeNote 4).


11. Protein assay kit (Lowry test, e.g., available from BIO-RAD).


12. 96-Well plate (with transparent bottom).
    Evaluation of radioactive L-phenylalanine incorporation into
    proteins


13. Cold trichloroacetic acid (TCA): 100% (w/v) trichloroacetic
    acid in H 2 O.


14. Cold NaOH: 100 mM NaOH.


15. Cold TCA: 5% (v/v) TCA in H 2 O.


16. Formic acid: 26.5 M formic acid (store at room temperature).


17. Scintillation liquid (e.g., Ultima Gold, PerkinElmer) (store at
    room temperature).


18. 20 ml vials for scintillation counter.


19. Scintillation counter.

2.3 Evaluation
of p70S6K and eEF2
Signaling Pathway

SDS polyacrylamide gel electrophoresis (SDS PAGE)

1. Sodium dodecyl sulfate (SDS): 5% (w/v) SDS in H 2 O.


2. Running gel buffer: 1 M Tris–HCl, pH 8.8.


3. Stacking gel buffer: 0.25 M Tris–HCl, pH 6.8.


4. Acrylamide/bisacrylamide solution: 30% acrylamide/ bisacry-
   lamide (37.5:1 ratio).


5. Ammonium persulfate (APS): 10% (w/v) APS in H 2 O (store
   for long term at 20 C).
   6.N,N,N,N^0 -Tetramethylethylenediamine (TEMED, store at
   4 C).


6. Electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1%
   (w/v), SDS.


7. 4loading buffer: 200 mM Tris–HCl, pH 6.8, 40% (v/v)
   glycerol, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue,
   25% (v/v)β-mercaptoethanol.


8. Molecular weight markers: PageRuler Prestained Protein
   Ladder.
   Immunoblotting


9. Blotting buffer: 25 mM Tris–HCl, 192 mM glycine.


10. Polyvinylidene fluoride (PVDF) membrane: 0.45μm pore size.

Study of Cardiac Hypertrophy and Protein Synthesis 325