Animal
Holstein, female, 65months
Muscle
masseter, semitendinosus, 0 day postmortem
(Muscle were excised from the cow and frozen in liquid nitrogen within 1h after slaughter.)
Sample preparation
<Whole protein>
Muscle powder was homogenized in 20 × volume (v/w) lysis buffer (8M urea, 30mM Tris-HCl, 4% (w/v) CHAPS, pH8.5). The homogenate was shaken for 30min. Then the homogenate was centrifuged at 12000 × g, 4℃ for 30min to pellet insoluble material. The supernatant was recovered.
<Myofibrillar protein>
Muscle powder was homogenized in 10 × volume (v/w) isolating buffer (20mM potassium-phosphate buffer(pH6.8) containing 100mM KCl, 1mM MgCl2, 1mM EDTA, 1mM 2-mercaptoethanol, and 0.05% sodium azide). In all steps of the myofibril preparation, the samples were kept on ice. The homogenate was washed twice in 5 × volume (v/w) isolating buffer and then was washed twice in 5 x volume (V/W) distilled water by centrifuging at 1000rpm, 4℃ for 15min. The pellet was solubilized in lysis buffer (8M urea, 30mM Tris-HCl, 4% (w/v) CHAPS, pH8.5).
<Sarcoplasmic protein>
Muscle powder was homogenized in 20 × volume (V/W) 40mM Tris-HCl (pH7.5). The homogenate was centrifuged at 12000 × g, 4℃ for 30min to pellet insoluble material. The supernatant was recovered.
Protein labeling
Muscle protein was labeled by CyDye DIGE Fluor minimal dyes (GE Healthcare, UK).
2D-electrophoresis
<First dimensional isoelectric focussing (IEF)>
Apparatus: Protean IEF cell (Bio-Rad, USA)
Protein loaded: 25µg each
Immobilised pH gradient (IPG) strip: 11 cm, pH 3-10 (Bio-Rad, USA)
Focusing condition:
step1: 250V, 15min
step2: 8,000V, 2.5h
step3: 8,000V, 35,000 Vh
<Second dimensional SDS-PAGE>
Apparatus: Criterion cell (Bio-Rad, USA)
Gel: 12.5% gradient PAGE gel (Bio-Rad, USA)
Condition: 200V for 55min
Mass Spectrometry
In-gel digestion: trypsin
Matrix: α-cyano-4-hydroxycinnamic acid
Apparatus: MALDI-TOF MS (REFLEX II, Bruker Daltonics, Bremen, Germany)
Ion detection: reflectron mode
Voltage: acceleration voltage of 20 kV
Pulse Extraction: delayed extraction
Search engine: MASCOT (Matrix Science, London, UK)
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