Gamma globulin

The content of sarcoplasmic (water-soluble) and myofibrillar (salt-soluble) proteins was determined as per the method of Hultmann and Rustad [15 (link)] with slight modification. A ground sample (4 g) was mixed with 80 mL of 0.05 M phosphate buffer (pH 7.0) and homogenized for 1 min using an IKA Labortechnik homogenizer (Selangor, Malaysia) at a speed of 9500× g. The homogenate was then centrifuged at 10,000× g for 20 min (4 °C) using a refrigerated centrifuge (Beckman Coulter, Avanti J-E Centrifuge, Palo Alto, CA, USA). The supernatant was collected, and the volume was adjusted to 100 mL with phosphate buffer (water-soluble fraction). The remaining precipitate was further homogenized for 10 s in 0.05 M phosphate buffer containing 0.6M KCl and 0.5% tritonX-405 (pH 7.0), followed by centrifugation as described above. The supernatant was collected and adjusted to 100 mL with KCl-phosphate buffer (salt-soluble fraction). Protein contents in the water- and salt-soluble extracts were determined using the BioRad protein assay with gamma globulin as a standard and expressed as a percentage (%) of the wet weight sample.

SEC was performed on a HiPrep Sephacryl S-200 high-resolution column run on an AKTA explorer FPLC. The column was equilibrated and eluted at a rate of 1 ml/min with a buffer containing 200 mM NaCl, 25 mM Tris (pH 8.0), and 2 mM DTT. Purified CHIL proteins with the His tags removed were used for analysis. Molecular weight reference standards were generated using vitamin B12, myoglobin (horse), ovalbumin (chicken), gamma globulin (bovine), and thyroglobulin (bovine), available as a broad molecular weight marker reference set (Bio-Rad).