Trans blot transfer cell

Purified rCsHK (2 µg) and total worm extract (30 µg) were subjected to 12% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Whatman, UK) at 100 V for 1 h in a Trans-Blot transfer cell (Bio-Rad, USA). The PVDF membranes were blocked with 5% (w/v) skimmed milk in phosphate buffer saline (PBS, pH 7.4) overnight at 4°C and then probed with a mouse anti-His tag monoclonal antibody (1∶2,000 dilution, Novagen, USA), mouse anti-rCsHK serum (1∶2,000 dilution) or serum from a pre-immune mouse (1∶2,000 dilution) for 2 h at room temperature. After washing with PBS 3 times, the membranes were incubated in horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1∶2,000 dilution, Protein tech., USA) for 1 h at room temperature. Both the primary and secondary antibodies were diluted with 0.1% BSA in PBS (pH 7.4). After washing 5 times, the membranes were finally developed in color with diaminobenzidine (DAB, Boster, China) reagents according to the manufacturer’s instructions.

SDS-PAGE and Western blot were performed as previously described (13 (link)). Briefly, cells were lysed in RIPA buffer [100 mM tris(hydroxymethyl)aminomethane (Tris)–HCl pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM MgCl₂] in the presence of a complete protease-inhibitor mixture. Protein content was determined by the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA). Cell lysates (30 µg/ml) were loaded onto SDS-PAGE and, after electrophoresis, proteins were transferred onto nitrocellulose membrane (GE Healthcare, Pittsburgh, PA, USA) by means of a Trans-Blot transfer cell (Bio-Rad Laboratories). The membranes were then blocked in 5% nonfat milk and incubated with the appropriate antibodies in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Anti-ERβ mAb (clone CWK-F12 from DSHB) was used as primary Ab. Peroxidase-conjugated goat anti-mouse IgG was used as secondary Ab (Bio-Rad Laboratories) and the reactions were developed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). To ensure the presence of equal amounts of protein, the membranes were reprobed with a rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase Ab (Sigma). Quantification of protein expression was performed by densitometry analysis of the autoradiograms (GS-700 Imaging Densitometer, Bio-Rad Laboratories).

ESPs or crude antigens of C. sinensis adult worms were subjected to 12% SDS-PAGE, then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) at 100 V for 1 h in a Trans-Blot transfer cell (Bio-Rad, USA). The membrane was incubated with an anti-rCs1 monoclonal antibody (mAb; Cs1-2-6-3) at 1:200 dilution, and subsequently in horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; Sigma, USA). Finally, the color was developed with diaminobenzidine substrate solution.