Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Nuances of electrophoresis study of titin/connectin

“…Based on data obtained the assumption on the existence of isoforms of T1 was made Horowits 1992). This assumption is in agreement with the results of our experiments (Vikhlyantsev and Makarenko 2000) in which the differences in electrophoretic mobility of T1 in striated muscles of a ground squirrel were found in a 7% polyacrylamide gel, prepared according to Fritz et al (1989). The distinguishing feature of an acceptable gel for highmolecular-mass proteins (greater than 500 kDa) is the requirement for a low concentration of bis-acrylamide (bis-acrylamide/acrylamide = 1:200) (Table 1, Reference 5).…”

“…Leupeptin, E-64, and a protease inhibitor cocktail (Sigma) have been used to inhibit proteolytic degradation of titin (Granzier and Irving 1995;Tatsumi and Hattori 1995;Linke et al 1997;Neagoe et al 2003;Warren et al 2003a;Vikhlyantsev and Podlubnaya 2006). In order to attain better solubilization of titin, it has been proposed to use urea-thiourea SDS DTT sample buffer (Fritz et al 1989;Warren et al 2003a). It is conceivable that urea-thiourea may facilitate the access of SDS to titin by rapidly solubilizing other myofibrillar proteins in the sarcomere lattice.…”

“…To prevent disulfide crosslinking, Fritz et al (1989), as well as Warren (2009, 2012), recommended the inclusion of β-mercaptoethanol in the top anodic buffer. Granzier and Wang (1993), using 3.3-12% linear gradient gels, noted the importance of polymerizing the gel for at least 12 h prior to sample application.…”

Effect of White Striping on Chemical Composition and Nutritional Value of Chicken Breast Meat

“…A sample (0.5 mL) of this homogenate (P1) was saved and the centrifugation repeated. This process was repeated to obtain S1 up to S4 and P1 up to P4 (Fritz et al, 1989). A composite sample from S1 to S4 was used for sarcoplasmic fractions, whereas P4 was used for myofibrils.…”

“…A composite sample from S1 to S4 was used for sarcoplasmic fractions, whereas P4 was used for myofibrils. Samples were mixed 1:1 with standard sample buffer which contained 8 M urea, 2 M thiourea, 3% (w/v) SDS, 75 mM Dl-dithothreitol, and 25 mM Tris HCl at pH 6.8 (Fritz et al, 1989), heated at 100°C for 5 min in a water bath, cooled, and applied to the gel. Fifteen µL of myofibrillar protein extract were loaded on 12% Mini-PRO-TEAN ® TGX Stain-Free™ Gel (BioRad, Hercules, CA, USA) and the same amount of sarcoplasmic extract was loaded on Mini-PRO-TEAN ® TGX any kD Stain-Free™ (BioRad).…”