Mini protean system

Western blotting was performed to verify the cross-reactivity of rabbit anti-human PGP9.5 (7863-2004, Bio-Rad, Hercules, CA, USA) to the PGP9.5 present in donkey testes according to the previously reported protocol with minor modifications [2 (link),11 (link)]. The testicular samples, which had been stored at −80 °C, were thawed at room temperature and homogenized using a Polytron PT 1200 CL homogenizer (Kinematica AG, Littau, Lucerne, Switzerland). Sample preparation buffer (0.5 M Tris-HCL (pH 6.8), 0.1% glycerol, 10% sodium dodecyl sulfate (SDS), 0.05% 2-β-mercaptoethanol, and bromophenol blue in distilled water) was used to dilute the concentration of quantified protein to 2 mg/mL. After being heated in a boiling water bath for 15 min, 15-μL samples were loaded onto a 10% SDS-polyacrylamide gel and separated using a Mini-Protean system (Bio-Rad, Hercules, CA, USA). The samples were electrotransferred to a polyvinylidene difluoride membrane (Millipore) and blocked with skim milk (BD Biosciences, San Jose, CA, USA). The membrane was incubated with PGP9.5 antibody diluted to 1:500 in skim milk overnight at 4 °C. For the negative control, the membrane was treated with normal mouse immunoglobulin G (IgG) at the same concentration of primary antibody. Horseradish peroxidase-conjugated anti-mouse IgG diluted in skim milk (1:10,000) was used as a secondary antibody.

To assess C6 on a protein level, rabbit serum was separated by SDS-PAGE (Mini-Protean- System, Biorad). The gel (12%) was directly stained with Coomassie (loading control) or used for blotting. After electroblotting onto a nitrocellulose membrane and blocking in 5% BSA (1 h, RT), membranes were incubated with a C6-specific monoclonal antibody (WU6-4; 1:200, Hycultec, Beutelsbach, Germany) overnight at 4 °C. C6 was visualized using an ECL Plex goat-α-mouse IgG-Cy5 (1:1000, Amersham, GE Healthcare, Freiburg, Germany).

The crude barley extract (cBP, at a concentration of 1 mg/mL) and its fractions eluted by DEAE ion-exchange chromatography (15 μL/per lane) were subjected to SDS- PAGE according to the standard protocol used in the laboratory [15 (link)]. Briefly, the proteins were separated on gels (15% for cBP and 12.5% for fractions), under a constant current of 30 mA, using a Mini-PROTEAN system (Bio-Rad Laboratories, Inc., Warsaw, Poland). The gels were stained with 0.1% Coomassie Brilliant Blue R-250 dye (Sigma-Aldrich, Poznań, Poland). We used Sigma Markers in the range of 6.5 - 66 kDa (Sigma-Aldrich, Poznań, Poland) to estimate the proteins’ molecular weight profile in cBP and barley protein fractions.

Binding reactions (10 μl) contained 40 mM Tris-HCl, pH 7.6, 1 mM DTT, 0.4 mM EDTA, 1–2% glycerol, 50 mM NaCl, 10 mM MgCl₂, 500 μg/ml bovine serum albumin (BSA), 2 nM of 5′-labeled DNA and varying concentrations of MgaSpn-His (20 to 180 nM). Reactions were incubated at ambient temperature for 20 min. Free and bound DNA forms were separated on native polyacrylamide (5%) gels (Mini-PROTEAN system, Bio-Rad) using Tris-borate-EDTA, pH 8.3, buffer (TBE). Gels were run at 100 V and ambient temperature. Labeled DNA was visualized using a Fujifilm Image Analyzer FLA-3000 and quantified using the Quantity One software (Bio-Rad).
For competitive EMSA, a 407-bp DNA fragment from the 5′ untranslated region of the hlyR gene was generated by PCR using primers Hly Sal/Eco5 and Hly Sal/Hind3 (Table 1). For each reaction, 50 ng of the pneumococcal 222-bp DNA and 150 ng of the 407-bp DNA (competitor DNA) were mixed with increasing concentrations of H-NS-His in binding buffer (250 mM HEPES, pH 7.4, 350 mM KCl, 5 mM EDTA, 5 mM DTT, 500 μg/ml BSA, 25% glycerol) and incubated at 37°C for 30 min. Samples (20 μl) were loaded onto native polyacrylamide (5%) gels (TBE buffer). Bands were visualized using a Gel-doc system (Bio-Rad).

Electrophoresis (SDS-PAGE) was performed in the Mini-PROTEAN system (Bio-Rad, Hercules, CA, USA), using 4% polyacrylamide stacking gels and 12.0% polyacrylamide resolving gels in reducing conditions and standard Tris-glycine buffers [24 (link)]. The image was obtained using a Gel Doc Documentation System (Gel Doc™ XR+ System, Bio-Rad Laboratories Inc., Hercules, CA, USA).