, 50 mL tubes. 2. 10, 100, and 1,000 mL pipettes

, Spectrophotometer cuvettes

, 25 mL syringe, type Microliter (Hamilton)

, Nitrocellulose membrane with 0.45 mm pore sizes (Schleicher & Schuell)

, Distillated water (ddH 2 O) (see Note 1)

. Np-40,

, Sodium deoxycholate (C 24 H 39 NaO 4 )

, Sodium orthovanadate (Na 3 VO 4 )

7. Ethanol,

, Absolute methanol

, Isopropanol. 8. Filtered sodium dodecyl sulfate (SDS) 10% (w/v)

. Tris, Trizma-base): powder form and prepared solutions (Tris-HCl 1.5 M-pH 8.8, Tris-HCl 1 M-pH 7.4 and 0.5 M

, We use commercially available acrylamide:Bis solution (BioRad) containing 29:1 acrylamide:Bis. The solution can be stored at 4°C for 1 month

, Prepare and store aliquots at ?20°C to avoid repeated freeze/ thaw cycles

, pH of Tris solution may change, especially during the first days following its preparation. If you see any impurity, discard the buffer and don't hesitate to prepare a new one

, Overlaying the gel with water or isobutanol prevents contact with atmospheric oxygen (which inhibits acrylamide polymerization) and helps to level the resolving gel solution

, Prepare the SDS extraction buffer by mixing 1 mL of 10% SDS, 100 mL of sodium vanadate 100 mM

, mL of ddH 2 O. Protease cocktail inhibitor (Roche) should be prepared according to manufacture instructions (1 tablet/2 mL of ddH 2 O)

, Prepare RIPA buffer by mixing 500 mL of Tris-HCl 1 M, 300 mL of NaCl 5 M, 100 mL of NP-40, 100 mL of SDS, 250 mL of sodium deoxycholate, and 200 mL of protease cocktail inhibitor

, Prepare Laemmli buffer (2×) by mixing 125 mL of Tris 0.5 M, 200 mL of SDS, 200 mL of Glycerol, 2 mg of bromophenol Blue, 50 mL of b-mercaptoethanol, 1 mL of 1 M DTT, and 424 mL of ddH 2 O

, 192 mM glycine). Weight 30.3 g Tris and 144 g glycine, mix, and make it to 1 L with ddH 2 O. Dilute 100 mL of 10X native buffer to 990 mL with ddH 2 O and add 10 mL of 10% SDS. Care should be taken to add SDS solution last, Prepare 10× native buffer (0.25 M Tris

, Dilute 100 mL of 10× native buffer to 990 mL with ddH 2 O. Add 150 mL methanol and 1 mL of 10% SDS

, Overlay the resolving gel with isobutanol prevents contact with atmospheric oxygen (which inhibits acrylamide polymerization) in addition to helping to level the resolving gel solution

, In some cases, especially when working in a hot area, the polymerization process may be fast and addition of isopropanol may result in a curved line

, Centrifuging the samples prior to the run helps remove non soluble debris

, Cut a tiny wedge from the bottom left side of the marker lane and the main membrane for orientation purposes

, One can also use PVDF membrane. In this case an additional step consisting of its activation in methanol is necessary

F. Djouadi, Y. Lecarpentier, J. L. Hebert, P. Charron, J. Bastin et al., A potential link between peroxisome proliferator-activated receptor signalling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy, Cardiovasc Res, vol.84, pp.83-90, 2009.

N. Salvi, A. Guellich, P. Michelet, A. Demoule, L. Guen et al., Upregulation of, 2010.
URL : https://hal.archives-ouvertes.fr/inserm-02426556

, PPARbeta/delta is associated with structural and functional changes in the type I diabetes rat diaphragm, PLoS One, vol.5, p.11494

P. Arck, B. Toth, A. Pestka, and U. Jeschke, Nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family in gestational diabetes: from animal models to clinical trials, Biol Reprod, vol.83, pp.168-176, 2010.