Mini protein 2 electrophoresis unit

Firstly, 27 mL of 5% SDS solution (95 °C) was added to surimi paste or gel sample (3 g) and homogenized using an IKA T25 digital ULTRA-TURRAX homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany) at 6000 rpm to solubilize the samples. To dissolve the sample, the homogenate was then incubated for 60 min at 95 °C. The protein sample solution was separated from undissolved matter by centrifuging using a Model Allegra 25R centrifuge (Beckman Coulter, Palo Alto, CA, USA) at 6050× g for 20 min at 25 °C. A biuret test was conducted to determine the protein concentration of the protein sample [16 (link)]. SDS-PAGE was performed as described by Laemmli [17 (link)]. SDS-PAGE consisted of 10% running gel and 4% stacking gel was used. A Miniprotein II electrophoresis unit (Bio-Rad Laboratories, Richmond, CA, USA) was used for electrophoresis at a constant current of 15 mA/gel. After separation, the procedure outlined by Quan and Benjakul [10 (link)] was adopted for staining and destaining the protein bands.

The fermentation solution and purified HMG fusion proteins from different purification processes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with 8% acrylamide gel and 5% condensing gel in the Mini-Protein II electrophoresis unit (Bio-Rad, USA) and stained with 0.25% Coomassie brilliant blue R-250 (Aldrich, USA).
IEF was used to predict the isoelectric point (pI) of HMG. In a separate set of experiments, 2 µg of purified HMG, mG-CSF, HSA and a mixture of HSA, mG-CSF and HMG prepared in 20 mM PB (phosphate buffer, pH 7.4) were loaded and analyzed on a Pharmacia MultiphorII horizontal electrophoresis system (GE Healthcare, USA) using ampholine, pH 3.5–10 (GE Healthcare, USA).
These samples were also analyzed using size exclusion chromatography on a TSK-GEL G3000SW columns (7.5×300 mm) (Tosoh, Japan) at a flow rate of 0.6 ml/min in 20 mM sodium phosphate (pH 7.5) and 0.15 M NaCl. The absorbance was monitored at 280 nm.

The cellulolytic microorganism isolates were used to inoculate 100 mL liquid medium C(CMC-Na 10 g, peptone 10 g, NaCl 5 g and 1000 mL water, adjust the pH to 7.0) at 37 °C. After 30 hours of culture, the broth was separated by centrifugation (8,000 g, 10 min) at 4 °C and the supernatant was collected. The purity of the cellulases was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% acrylamide gel and 5% condensing gel in a Mini-Protein II electrophoresis unit (Bio-Rad). The gel was stained with 0.25% Coomassie brilliant blue R-250 (Aldrich, USA). Running and staining procedures were performed according to the supplier’s protocol^{34 (link)}. The protein concentration was determined by the Bradford method with bovine serum albumin as a standard^{34 (link)}.