Protein molecular weight marker

DNA purification kit was obtained from PEQLAB. PCR Purification Kit was purchased from Bioneer (Seoul, Korea). Plasmid purification kit was purchased from Roche (Basel, Switzerland). TAclone PCR Cloning Kit was provided by insta-clone kit. DNA ladder, Protein molecular weight markers, Taq polymerase, Pfu DNA Polymerase, T4 DNA ligase, XhoI and NcoI enzyme, IPTG, and X-GAL were purchased from Fermentas (Glen Burnie, MD, USA). Ampicillin, kanamycin, and 2,6-dimethylphenol (DMP) were obtained from Sigma (St. Louis, USA). E. coli DH5α and E. coli BL21 (DE3) strains, and vector pET-26b (+) were obtained from Invitrogen (Carlsbad, CA, USA). Ni Sepharose resin was purchased from Qiagen. All other chemicals were provided from Merck (Darmstadt, Germany).

SDS-PAGE was performed by the procedure of Laemmli using 10–12% polyacrylamide gels in presence of 5% 2-mercaptoethanol in sample buffer. Protein molecular-weight markers (Fermentas or Biorad) were used as MW standards. Proteins were visualized by Coomassie brilliant blue staining. Western blotting was performed on nitrocellulose filter (Biorad). Blots were immunostained with 100 times diluted mouse anti-SjE16.7 serum, and 1000 times diluted horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Cell Signaling). Enhanced chemiluminescence (ECL, Pierce) was used as substrates and signals were analyzed by Luminescent imager (ImageQuant Las 4000, GE Healthcare).

PAGE was performed according to Laemmli [48 (link)] with modifications (Laemmli buffer additionally contained 5% β-mercaptoethanol and 0.625 mM EDTA) in 4% concentrating and 12% resolving polyacrylamide gel [13 (link)]. Various CW extracts were equalized by the optical density of CW at 540 nm. Cell lysates were equalized by the amount of proteins according to Lowry [47 (link)]. PAGE was performed in the presence of prestained protein molecular weight markers (Fermentas, Moscow, Russia). Protein staining in gel was performed with Coomassie G-250 according to Peisker [49 (link)] or with silver nitrate according to Gharahdaghi [50 (link)] with modifications. To identify Bgl2 bands Western Blot analysis was performed according to Rekstina [22 (link)] with modifications. Primary polyclonal antibodies against Bgl2 were raised in male BALB/c mice (SPF status) in the laboratory of Dr. O.S. Morenkov (Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia) with PAGE-purified protein (40 μg per mouse) and were used in previous investigations [12 (link),13 (link),22 (link)]. Secondary polyclonal rabbit anti-mouse IgG antibodies were labeled by horse radish peroxidase (Invitrogen, Moscow, Russia). Protein–antibody complexes were visualized by enhanced chemiluminescence using the ThermoFisher Scientific ECL system (ThermoFisher Scientific, Moscow, Russia).

The purified MAG protein was separated by SDS-PAGE to identify the protein molecular weight through protein molecular weight marker (ThermoFisher scientific) and analyzed by western blot. The Immobilon-PSQ PVDF membrane (0.2 μm pore size, Millipore, USA) onto which purified protein was transferred was incubated with pig anti-T. gondii-positive or -negative serum and then detected with horseradish peroxidase (HRP)-conjugated rabbit anti-swine IgG (H+L) (Frdbio Bioscience & Technology, China).

Urinary glycoproteins were further analyzed using lectin blotting. Firstly, the pooled urinary proteins were analyzed by SDS-PAGE. Briefly, urinary proteins were mixed with 5 × loading buffer and boiled for 5 min at 100°C; after that, the samples were spun down and loaded on a 10% polyacrylamide resolving gel and a 3% stacking gel, and protein molecular weight marker (Thermo Scientific, Waltham, USA) was run in gel simultaneously. After being immobilized, the gel was stained by alkaline silver. For lectin blotting analysis, the proteins in gels were transferred to a PVDF membrane (Millipore, MA, USA) at 100 V for 90 min in wet transfer device (Beijing Liuyi Instrument Factory, China). Subsequently, the membranes were washed twice with TBST buffer (150 mM NaCl, 10 mM Tris-HCl, and 0.05% v/v Tween-20, pH 7.5) and blocked for 1 h with Carbo-Free Blocking Solution (Vector, Burlingame, CA) for 1 h at ambient temperature. The membranes were then incubated with 3 µg/ml Cy5 (GE Healthcare, Biosciences, Piscataway, NJ, USA) labeled lectins in previous blocking solution with gentle shaking overnight at 4°C below protection from light. The membranes were washed twice each for 10 min with TBST and scanned by Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) in red fluorescence channel 635 nm excitation/650LP emission.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified PPO from O. ficus-indica was applied according to the Laemmli method [25 (link)]. The purified PPO of O. ficus-indica was subjected to SDS-PAGE in a Mini Protean Tetra Cell Electrophoresis Unit (Bio-Rad Laboratories, Hercules, CA, USA), with 3% stacking gel and 10% running gel. The purified PPO by affinity gel from O. ficus-indica and standard proteins were run on the SDS-PAGE gel. The purified PPO and marker were loaded in lanes, and the slab gels were 1 mm thick. The PPO and marker were run at 80 V in the stacking gel and 150 V in the separating gel (running gel). After the electrophoresis, the protein bands were visualized using Coomassie Brilliant Blue R-250. The protein molecular weight marker (Thermo Scientific, Lithuania), which was used for comparison to the molecular weight, included lysozyme (14.4 kDa), β-lactoglobulin (18.4 kDa), REase Bsp98l (25.0 kDa), lactate dehydrogenase (35.0 kDa), ovalbumin (45.0 kDa), bovine serum albumin (66.2 kDa), and β-galactosidase (116.0 kDa).

For separating rPLD1 and mutant-rPLD1, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was applied according to Laemmli et al. [40 (link)]. For preparation of protein samples, rPLD1, mutant-rPLD1 and bacterial lysate supernatant were separately mixed with sample buffer, 15–20 µg of each sample subjected to SDS-PAGE. All samples were then heated at 95 °C for 5 min. Thereafter, each sample was pipetted into one of the wells in the gel. Also protein molecular weight marker (Thermo Fisher Scientific Co. Waltham, MA, USA.) was loaded into its own well in the gel. Then, a 15-mA electrophoretic flow was applied to the gel for initial 30 min, followed by 25-mA for 2 h in order to migrating of negatively charged proteins through the gel in the direction of anode pole. Finally, the gel was brought out of the glass plates and placed in the Coomassie Brilliant Blue^® R-250 (Thermo Fisher Scientific Co. Waltham, MA, USA.) solution for staining for 12 h at room temperature followed by immersing gel into the destaining solution until background of the gel was fully destained and protein bands became visible.