Electrophoresis of shrimp basic proteins

Description

This platform allows basic proteins to be analyzed by two-dimensional polyacrylamide gel electrophoresis.

Brief protocol

The pellet of ribosomal proteins were resuspended in sample buffer（8 M urea, 40 mM Tris-HCl, 2.3 mM EDTA, 0.06 % TEMED, 52 mM boric acid, 5% BME）and analysed by basic-urea polyacrylamide electrophoresis system. The first dimension of electrophoresis was carried out in tube gel（130 × 2.5 mm）containing 8%〔w/v〕 acrylamide, 0.3%〔w/v〕bis-acrylamide, 6 M urea, 21 mM EDTA, 520 mM boric acid, 400 mM Tris-HCl, 0.1 % 〔w/v〕ammonium persulfate, and 0.1 %〔w/v〕 TEMED at pH 8.6 in the running buffer （6.5 mM EDTA, 156 mM boric acid, 120 mM Tris-HCl, pH 8.6）at 150 V for 24 h. After the first dimension electrophoresis was completed, the gel was extruded from glass tube and incubated in the equilibration buffer（0.05 M Tris-HCl, pH 6.8, 10 % 〔v/v〕 glycerol, 2 %〔w/v〕SDS, 5 %〔v/v〕BME, and 0.1 % 〔w/v〕bromphenol blue ）for 15 min with twice. Then, put the gel into the second-dimension gel, usually 15 %, and sealed with agarose. The second-dimension electrophoresis（Mini-PROTEIN II, Bio-Rad）was carried out at 150 V for 6 h. Gels were stained with SYBRO Ruby for 24 h and scanned with fluorescence scanner (Typhoon 9200) at 520 nm for image analysis.