- Transfection reagent. Store at 4C.
- 5 siRNA buffer (Dharmacon, Cat #B-002000-UB-100).
Store at 4C. Dilute sterilely 1:5 with RNase-free water to
1 siRNA buffer for usage. - siRNA oligo pools: 20μM stock solution with 1siRNA
buffer. SMARTpool siGENOME (Dharmacon) used: Human
PRKAA1 M-005027-02 (siAMPKα1), human PRKAA2
M-005361-02 (siAMPKα2), and nontargeting siRNA pool
#2 D-001206-20 (siControl). Dilute sterilely and store at
20 C.
2.3 SDS
Polyacrylamide Gel
Electrophoresis
- Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8, 0.4% (w/v)
SDS in H 2 O. - Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8, 0.4% (w/v) SDS
in H 2 O. - Acrylamide 4 K solution (30%) -Mix 37.5:1.
- APS: 20% (w/v) ammonium persulfate (APS) in H 2 O. Store
long term at 20 C and short term at 4C. - N,N,N,N^0 -Tetramethylethylenediamine (TEMED). Store at
4 C. - Isopropanol.
- PBS. Store at 4C.
- Frackelton buffer: 20 mM HEPES, pH 7.4, 50 mM NaCl,
30 mM Na 4 P 2 O 7 , 50 mM NaF, 0.2% (v/v) Triton®X-100,
10% (v/v) glycerol, 5μM ZnCl 2 , Protease Inhibitor Cocktail
(PIC), 20 mMβ-glycerophosphate, 1 mM vanadate (Na 3 VO 4 ),
50 nM okadaic acid. Store at 4C(seeNote 1). - Lowry protein assay kit.
- Laemmli buffer: 25 mM Tris–HCl, pH 8.3, 250 mM glycine,
0.1% (w/v) SDS. - 4loading buffer: 320 mM Tris–HCl, pH 6.8, 40% (v/v)
glycerol, 20% (w/v) SDS, 0.5% (w/v) bromophenol blue,
200 mMβ-mercaptoethanol. - Size marker: prestained protein marker (10–250 kDa range).
- Polyacrylamide protein (mini-)gel system with 1 mm spacer
glass plates.
2.4 Immunoblotting 1. Semidry transfer buffer: 25 mM Tris base, 192 mM glycine,
20% (v/v) methanol.
- Tank blot transfer buffer: 25 mM Tris base, 192 mM glycine,
0.01% (w/v) SDS. Store at 4C. - Nitrocellulose membrane: 0.2μm pore size.
- Grade 3 Qualitative Filter Paper.
376 Sarah Krieg et al.