Model vcx 500 ultrasonicater

Total proteins were extracted from jejunum mucosal scrapings by following an existing procedure with slight modifications (Wang et al. 2009 (link)). In brief, frozen samples of jejunum mucosal scrapings from all pigs in two groups were crushed in a mortar containing liquid nitrogen. The powder (approximately 100 mg per sample) was transferred to sterile tubes with lysis buffer (LB; containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v DTT, 1% v/v IPG Buffer pH 4–7, 1% v/v proteinase inhibitor cocktail). The mixture was sonicated in an ice bath using a Model VCX 500 Ultrasonicater (Sonics & Materials, Newtown, CT, USA) at 20% power output for 10 min with 2-s on and 4-s off cycles. Subsequently, the lysed cell suspension was incubated at room temperature for 1 h to solubilize proteins. After centrifugation at 40 000 g and 4 °C for 40 min, the supernatant protein was collected and its protein concentration was determined according to a modified Bradford assay (Ramagli & Rodriguez 1985 (link)). The protein concentration was 6.84±0.42 mg/ml.

Protein extraction was conducted as described with slight modifications [55 (link)]. In brief, frozen samples of liver from all pigs in three groups were crushed in a mortar containing liquid nitrogen. The powder (approximately 100 mg per sample) was transferred to sterile tubes with 1mL lysis buffer (LB; containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v DTT, 1% v/v IPG Buffer pH 4–7, 1% v/v proteinase inhibitor cocktail). The mixture was sonicated in an ice bath using a Model VCX 500 Ultrasonicater (Sonics & Materials, Newtown, CT, USA) at 20% power output for 10 min with 2-s on and 4-s off cycles. Subsequently, the lysed cell suspension was incubated at room temperature for 1 h to solubilize proteins. After centrifugation at 40,000× g and 4 °C for 40 min, the supernatant protein was collected and its protein concentration was determined according to a modified Bradford assay [56 (link)]. The protein concentration was 8.69 ± 0.94 mg/mL.