Materials and methods

Sample buffer. The sample buffer contained 8 M urea, 2M thiourea, 3% SDS (w/v), 75mM DTT, 0.03% bromophenol blue, and 0.05 M Tris–Cl, pH 6.8 (modified from [6]). Sample buffer (100ml) is prepared by adding solid urea and thiourea to a small volume of heated water (do not exceed 40 C due to rapid formation of cyanate) until dissolved completely. Then about 10g of mixed bed resin (TMD-8, Sigma M-8157) was added and the mixture was stirred for 15 min to deionize the urea. The slurry was then filtered through Whatman No. 1 filter paper to remove the resin. Tris-base, DTT, SDS, and bromophenol blue were then added and the pH was carefully adjusted to 6.8 using HCl (use special care near the end since the buffer capacity is very low); if you overshoot, use Tris-base to readjust. Sample buffer may be aliquoted and stored at) 20 C. Sample preparation. Cardiac tissue was dissected from Sprague–Dawley rats and Black Swiss mice and flash frozen in liquid nitrogen. The frozen tissue was pulverized and placed in preweighed 2-ml Dounce homogenizers. The weight of the tissue was determined and sample buffer was added (between 80: 1 and 200: 1 buffer to tissue v/w ratio). The tissue was dispersed using several strokes in the homogenizer. Samples were then vortexed thoroughly and heated at 100 C for 3 min. The samples were again vortexed and subsequently centrifuged for 5min at 13,200 g. The supernatant was removed, transferred to clean tubes, and either immediately loaded on the gels or stored at) 80 C prior to electrophoresis. Gel preparation. Glass plates were 16 x 18 cm, cleaned with soap and water, rinsed with 100% ethanol, and allowed to dry. After the glass plates were dried they were coated with Rain X or Sigmacote (Sigma) to allow for easier removal of the gels from the plates [DATD gels are stickier and more difficult to remove than biscross-linked gels]. A SE600 Hoefer gel system (Pharmacia) was used with 0.75-mm gel spacers. It is important to note that this large gel format is needed to adequately separate the isoforms. The following recipe is sufficient for one 16 x 18-cm gel. To make 17ml of a 6% T 2.6% C resolving gel, add 6.56 ml water, 3.4 ml 50% glycerol (v/v), 4.25 ml 1.5 M Tris, pH 8.8, 2.55 ml 40% acrylamide (37.5: 1 cross-linked with DATD (Bio-Rad)[7]), 170 ll 10% sodium dodecyl sulfate (w/v), 50 ll 10% ammonium persulfate (w/v), and 20 ll TEMED. The resolving gel was poured to allow a stacking gel with 1-cm deep wells and at least 1-cm length between the bottom of the wells and the resolving gel. Water was added to the top of the resolving gel to form a flat