Dye binding dc protein assay

The bacterial lysate used in the present study was prepared from the highly virulent Vh MML-1 strain of V. harveyi, as described previously [21 (link)], with minor modifications. Briefly, V. harveyi (Vh MML-1) was cultured in 50 ml of TSB with 2% NaCl at 25 °C to an OD₆₀₀ of 1. The bacteria were collected by centrifugation at 3000× g for 10 min and then washed three times with saline. Afterwards, bacteria were re-suspended in 2 mL saline, sonicated by using a VCX 130 ultrasonic processor (Sonics), and then centrifuged at 12,000× g for 30 min at 4 °C. The resulting soluble supernatant was used as the V. harveyi lysate. The lipopolysaccharide (LPS) in the V. harveyi lysate was removed by the Detoxi-Gel Endotoxin Removing column (Thermo Scientific) and its level (below 0.1 EU/mL) was confirmed by the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific) [22 (link)]. The protein concentration of the bacterial lysate was determined by using the dye-binding DC protein assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Aliquots of the bacterial lysate were stored at −20 °C until use.

The bacterial lysate was produced from the highly virulent Vh MML-1 strain of V. harveyi as described previously [8 (link),27 (link)]. V. harveyi (Vh MML-1) was grown in 50 mL of TSB with 2% NaCl at 25 °C to an OD₆₀₀ of 1. The cultured V. harveyi bacteria were spun down by centrifugation at 3000× g for 10 min and then washed three times with saline. Afterwards, bacteria were re-suspended in 2 mL saline, sonicated by an ultrasonic processor VCX 130 (Sonics, Newtown, CT, USA) and then centrifuged at 12,000× g for 30 min at 4 °C. The resulting soluble supernatant was collected to remove its residual lipopolysaccharide (LPS) by the Detoxi-Gel Endotoxin Removing column (Thermo Scientific, Waltham, MA, USA) and the level of LPS (below 0.1 EU/mL) was confirmed by the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, Waltham, MA, USA) [28 (link)]. The protein concentration of the bacterial lysate (soluble supernatant) was measured using the dye-binding DC protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as a standard. Aliquots of the bacterial lysate were stored at −20 °C until use.