, 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)
,
, Sodium deoxycholate (C 24 H 39 NaO 4 )
, Sodium orthovanadate (Na 3 VO 4 )
,
, Absolute methanol
, Isopropanol. 8. Filtered sodium dodecyl sulfate (SDS) 10% (w/v)
, 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
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. ,
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
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. ,