Gastrocnemius and quadriceps muscle samples were homogenized, and then lysed for 20 min in lysis buffer (iNtRON Biotechnology, Seoul, Korea) containing protease and phosphatase inhibitors. Lysate protein concentrations were evaluated in a BCA protein assay (Pierce, Rockford, IL, USA) using BSA as a standard. Equal amounts of proteins were separated by SDS-PAGE gels and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in 5% skim milk and then washed in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). The membranes were incubated overnight at 4°C with indicated primary antibodies. After a washing step, the membranes were blocked for 1 h in 5% skim milk containing peroxidase-conjugated anti-rabbit or anti-mouse. Signals were detected by EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and visualized using a LAS-4000 apparatus (GE Healthcare Life Sciences, Marlborough, MA, USA). Proteins band intensities on each blot were quantified by densitometric assessment using ImageJ software (Bethesda, MD, USA).
Las 4000 apparatus
Manufactured by GE Healthcare
The LAS-4000 apparatus is a laboratory equipment designed for scientific applications. It provides a controlled environment and automated functionality to support various experimental procedures. The core function of the LAS-4000 is to facilitate precise data collection and analysis within a regulated setting.
Automatically generated - may contain errors
2 protocols using las 4000 apparatus
1
Western Blot Protein Analysis
2
Western Blot Analysis of Muscle Proteins
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Quadriceps and gastrocnemius muscle samples were minced and lysed for 30 min in lysis buffer (iNtRON Biotechnology, Seoul, Republic of Korea) containing protease and phosphatase inhibitors. The protein concentration of each lysate was quantified using a BCA protein assay (Pierce, Rockford, IL, USA). Lysates containing equal amounts of proteins were subjected to SDS-PAGE and then transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). After 1 h of blocking in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 5% skimmed milk, the membranes were washed in TBS-T and incubated overnight at 4 °C with primary antibody. They were then washed and incubated in TBS-T containing 5% skimmed milk and secondary antibody (peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat antibody, Bio-Rad, Hercules, CA, USA). Specific protein bands were identified using an EZ-Western Lumi Femto kit (DoGenBio, Seoul, Republic of Korea) and imaged using an LAS-4000 apparatus (GE Healthcare Life Sciences, Marlborough, MA, USA). The relative band intensities were quantified using ImageJ 1.48 software (NIH).
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