Eba 1051

Polyacrylamide gel (12.5%) was used to perform sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Lee et al., 2020 (link)). The extracted myosin samples
(protein concentration, 0.5 mg/mL) were diluted twice (v/v) with sample
buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) and heated at 95°C.
The samples (0.5 mg protein/mL) and protein ladder (3454A, Takara Bio,
Shiga, Japan) were loaded at 10 and 5 μL, respectively. The bands
were stained with a solution containing Coomassie brilliant blue, acetic
acid, and methanol overnight. Then, a buffer containing acetic acid and
methanol was used to de-stain the gels. The gels were scanned at an optical
resolution (63.5 μm/pixel) with a densitometer (GS-710, Bio-Rad
Laboratories) and analyzed using Image Master 2D Platinum v5.0 (GE
Healthcare, formerly Amersham Biosciences, Seoul, Korea).

The myofibrillar protein extracts were diluted to a concentration of 2 mg/mL for
SDS-PAGE analysis. The myofibrillar protein samples were reacted with a sample
buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) at a ratio of 1:1 (vol/vol) at
90°C for 10 min. Electrophoretic separation was conducted using 12.5%
polyacrylamide gel in an electrophoresis system (AE-6531 mPAGE, ATTO, Tokyo,
Japan) and a protein ladder (3454A, Takara Bio, Shiga, Japan) was loaded to
figure out the molecular weights of protein bands. The protein bands were
stained using a solution containing Coomassie brilliant blue and de-stained
using a solution containing 10% acetic acid and 30% methanol. The SDS-PAGE gel
was scanned using Epson Perfection V850 Pro (V850 Pro, Epson, Long Beach, CA,
USA).

The digesta samples (1 mg/mL) were diluted with 2X sample buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) at a 1:1 (v/v) ratio and heated at 90 °C for 10 min in a heating block. Electrophoretic separation was conducted using 12.5% polyacrylamide gels. The protein bands were stained with Coomassie brillant blue buffer overnight and destained with acetic acid buffer. The gel was scanned using Epson Perfection V850 Pro (Epson, CA, USA).