Detection of myofibrillar proteins using a step gradient minigel with an ambiguous interface

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products

“…Kellermayer & Grama (2002) and Kurzban & Wang (1988). A comparable electrophoretic pattern of myofibrillar proteins has been reported previously by Chen et al (2005).…”

“…The addition of glycerol has been reported to facilitate gradient formation and also to improve resolution of the SDS-PAGE technique (Sobieszek, 1994). However, Chen et al (2005) did not find a great improvement of the gel system with the use of this reagent. Present research supports the first statement, given the fact that the addition of glycerol both to the resolving and the stacking gel solutions has successfully improved band resolution (comparison data not shown).…”

Decline in titin content in rat skeletal muscle after denervation

“…Also, the total content of protein in the myofibrils was measured with a protein assay kit (Bio‐Rad, Hercules, California), and the concentration was adjusted to 4 mg/ml with ice‐cold lysis buffer. Subsequently, myofibrils were solubilized with an equal volume of 2× SDS sample buffer (2 mM EDTA, 2% SDS, 1.2 M β‐mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, and 20 mM Tris‐base; pH 8.0) and heated to 50°C for 20 min 5…”

“…A step gradient minigel with an ambiguous interface was made by the improved method of Chen and coworkers 5. To determine the optical loading volume and assure working within the linear range of the system, a loading range of each sample was electrophoresed on a “calibration gel” as described previously 1, 33.…”

“…For immunoblot analysis, gels were electroblotted onto nitrocellulose paper 5. After blocking, the membrane was reacted with the primary antibodies of mouse anti‐titin (T12) monoclonal antibody (Boehringer Mannheim, Indianapolis, Indiana) and the primary antibody was omitted in the negative control.…”