Prestained sds page standard

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to separate purified rSAG1 protein on a 12% polyacrylamide gel along-with protein molecular marker as a standard (pre-stained SDS-PAGE standards, BioRad, Hercules, CA, USA). After electrophoresis, resolved proteins were transferred from gel to nitrocellulose membrane (Novex (USA), Invitrogen) which was then immune-blotted with anti-SAG1 antibodies or serum of T. gondii-infected mouse. The dilution of serum was made 1:200 in 5% skimmed milk-TNT (comprising 0.05% Tween-20, 140 mM NaCl, 15 mM Tris-HCl) [51] . Immunoblot was detected using anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) or anti-HIS antibodies (Invitrogen, Waltham, MA) with alkaline phosphatase. The dilution of anti-antibodies was made 1:1000 in 5% skimmed milk-TNT. The catalytic activity of alkaline phosphatase was detected by using liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA) i.e. 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium (BCIP/NBT).

Each sample (0.10 g) was solubilized in 1.0 mL of 9 M urea and an aliquot was added to a sample buffer containing Tris-HCl (0.6 M, pH 6.8), 2% SDS, 10% (vol/vol) glycerol, and 1.5% (wt/vol) DTT to obtain a final protein concentration of 4 mg/mL. Then, 10 μL of this solution was loaded on a 15% polyacrylamide pore gel (1.5 × 18 × 16 mm), and electrophoresis was carried out in a Hoefer SE 600 Series Ruby Standard Vertical Electrophoresis Unit (GE Healthcare BioSciences, Little Chalfont, UK), at 25 mA for 3 h at room temperature. The gels were stained with 0.25% (wt/ vol) CBB overnight (Candiano et al., 2004 ) and destained with water. Prestained SDS-PAGE standards (Bio-Rad, Richmond, CA) were used as protein molecular mass markers.

SDS-PAGE was done in 8% and 10% polyacrylamide separating gels (4% stacking gels) using a Mini-PROTEAN® Tetra vertical electrophoresis system and a PowerPac™ Basic Power Supply (BIO-RAD, Hercules, California, USA). Coffee enriched fractions (HMW F and LMW F), RCEs and GCEs were dissolved in Tris-HCl/SDS sample buffer (pH 6.8) containing 2-mercaptoethanol (50 g/L) and heated in a water bath for 3 min at 90 °C. In a second approach, electrophoresis was done under non-reducing conditions. Sample solutions (10 or 20 mg/mL, 16 μL) and a molecular weight marker solution (BIO-RAD prestained SDS-PAGE Standard, MW range 10-250 kDa,B10 μL) were each applied to a well.
After electrophoresis stacking at 25 mA and run at a constant voltage of 120V for 0.75 h, gels were silver stained according to the method of Heukeshoven and Dernick and interpreted.