Trans blot sd transfer cell

Protein samples including crude antigens of ML, IIL, AW and NBL, ES antigens of ML, and rTsNd were separated by SDS-PAGE using a 10% acrylamide separating gel and then transferred onto nitrocellulose membranes (Millipore, USA) using a trans-blot SD transfer cell (Bio-Rad, USA) [31 (link),32 (link)]. The membranes were cut into strips, blocked with 5% skimmed milk in Tris-Buffered Saline with Tween-20 (TBST) at 37°C for 1 h, and incubated at 37°C for 1 h with 1:100 dilutions of different mouse sera. After being washed, the strips were incubated at 37°C for 1 h with HRP-conjugated goat anti-mouse IgG (1:5000 dilution), and finally with 3, 3’-diaminobenzidine tetrahydrochloride (DAB; Sigma).

Cells, including HEK293T, MCF-7, MDA-MB-231, and MCF-12F with different treatments were washed with DPBS and incubated on lysis IP buffer (Thermo Fisher Scientific, 87787) on ice for 30 min. Cell debris was removed by centrifugation at 16,000 × g at 4°C for 8 min. The supernatants were obtained and mixed with 2x SDS sample buffer (Sigma-Aldrich, S3401), samples were then boiled for 5 min on a heat block. Next, proteins were separated using SDS-PAGE polyacrylamide gels (8–12%). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, 1620177) using a Trans-Blot SD transfer cell (BioRad, 1703940). Membranes were incubated for 1 h with blocking buffer (BioRad, 1706404) at room temperature, followed by an overnight incubation with primary antibodies (Table 1) at 4°C. Next, membranes were washed 3 times with PBS-tween (Sigma-Aldrich, P3563) followed by a 1-h incubation with the secondary antibodies (Table 1) at room temperature. Subsequently, three additional washes in PBS-tween were performed before imaging the membranes. Finally, membranes were developed by adding SuperSignal West Femto maximum sensitivity substrate (Pierce, 34095), and proteins were visualized in a Li-Cor Odyssey Fc Imager 2800 (Li-Cor, Lincoln, NE) and a ChemiDoc imaging system (BioRad, Hercules, CA).

To produce specific polyclonal antibodies against NT-OMV and HT-OMV sediment, New Zealand White rabbits (Envigo, Indianapolis, IN, USA) were immunized with 1 mg of the corresponding antigen in 0.2 mL of PBS, intramuscularly. Blood samples were collected before immunization and weekly until four weeks post-immunization. The presence of specific antibodies against bacterial vesicles in serum were determined by immunoblotting at week 0, 1, 2, 3, and 4 post-immunization. Immunoblotting was performed using nitrocellulose membranes (Whatman Protran^®; Merk kGaA, Darmstadt, Germany, pore size 0.45 µm) and a semidry blotting system at 0.8 mA/cm2 for 30 min (Trans-Blot^® SD Transfer Cell, BIO-RAD, Hercules, CA, USA). After that, protein-binding sites were blocked with PBS with 5% skimmed milk, overnight, at room temperature. Then, the membranes were washed 3 times with PBS-Tween and then incubated (4 h at 4 °C) with sera from hyperimmunized rabbits collected at week 0, 1, 2, 3, and 4 post-immunization. The membranes were washed 3 times with PBS-Tween and then peroxidase-conjugated secondary antibody (anti-IgGFc,) was added for 60 min at room temperature. Finally, membranes were washed with PBS-Tween and the antibody-antigen complexes were visualized after addition of a substrate/chromogen solution (H₂O₂/chloro α-naphthol).

The soluble protein fraction extracted after induction with IPTG of transformed, and non-transformed, M15 cells (see above), and the protein fractions purified by IMAC were separated in 10–20% linear gradient polyacrylamide gels, as previously described [33 (link)]. The separated proteins were stained with BlueSafe (Nzytech, Lisboa, Portugal) or transferred to PVDF membranes (Immobilon-P, Millipore Ibérica SA, Madrid, Spain) in a Trans-Blot SD transfer cell (Bio-Rad Laboratories, Richmond, CA, USA) and blocked with PBS with 0.05% Tween 20 and 1% dry skimmed milk (PBS-T-SM) for 2 h at RT on an orbital shaker. Membranes were then incubated with mAb US9 (1/2,000) followed by HRP-conjugated goat anti-mouse IgG antibody (Bio-Rad, 1/3,000) after a washing step (three times with PBS-T for 5 min each), or with HRP-conjugated mouse anti-polyhistidine (Sigma-Aldrich, 1/100,000). Each antibody was incubated for 1 h at RT on an orbital shaker. Finally, the membranes were washed again, and the bands were developed using 3,3-diaminobenzidine tetrahydrochloride tablets (Sigma-Aldrich), following the supplier’s instructions.

For the immunoblot analysis, 10 μg aliquots of each extract of F. hepatica, F. gigantica, C. daubneyi, D. dentriticum and S. mansoni were processed on 10–20% linear gradient polyacrylamide gels, under reducing conditions, following the procedure described by Laemmli [31 (link)]. The separated proteins were transferred to PVDF membranes (Immobilon-P, Millipore Ibérica SA, Madrid, Spain) in a Trans-Blot SD transfer cell (Bio-Rad) at 120 mA for 30 min. The membranes were subsequently blocked for 2 h at RT with PBS-T-SM and then incubated with mAb MF6 diluted 1:10,000 in PBS-T-SM for 1 h at RT on an orbital shaker. The membranes were washed three times for 5 min each with PBS-T and then incubated with HRP-labeled goat anti-mouse IgG secondary antibodies diluted 1/1,500 in PBS-T-SM for 1 h at RT. Finally, the membranes were washed again, and the bands were developed using 3,3’-diaminobenzidine tetrahydrochloride tablets (Sigma-Aldrich), following the supplier’s instructions.

To determine the antigenic characteristics of HT, immunoblotting assay was performed [30 (link)]. In brief, the components separated electrophoretically were transferred from the gel to a nitrocellulose membrane (Whatman Protran^®; Merck KGaA, Darmstadt, Germany, pore size 0.45 µm) using a semidry blotting system at 0.8 mA/cm² for 30 min (Trans-Blot^® SD Transfer Cell, Bio-Rad, Hercules, CA, USA). Protein-binding sites were blocked with 5% skimmed milk in PBS at room temperature overnight. Next, the membranes were washed three times with PBS-Tween and incubated with eight different sera from healthy donors or eight different sera from patients infected with ETEC, all diluted 1:80. After incubation at room temperature for 4 h, the membranes were washed three times with PBS-Tween and treated with peroxidase (PO)-conjugated secondary antibody GAHu/IgG (H+L), HRP conjugate (1:1000) or GAHu/IgA (Fc), HRP conjugate (1:1000) for 60 min at room temperature. Finally, membranes were washed with PBS-Tween and the antibody–antigen complexes were visualized by addition of a substrate/chromogen solution (H₂O₂/4-chloro-1-naphthol).

Cells were washed twice in phosphate-buffered saline and lysed in lysis buffer (10 mM sodium phosphate [pH 7.2], 150 mM NaCl, and 1% sodium dodecyl sulfate [SDS]) containing a protease-inhibitor cocktail (04080–24, Nacalai Tesque). Lysate proteins (10 μg) were separated by SDS-polyacrylamide gel electrophoresis. After transferring the proteins to a polyvinylidine difluoride membrane using a Trans-Blot SD transfer cell (Bio-Rad, 170–3940), mNeonGreen-3xFLAG and glyceraldehyde-3-phosphate dehydrogenase were recognized using the appropriate antibodies and developed with a chemiluminescent method according to standard protocols using SuperSignal West Dura Extended Duration Substrate (Pierce, 34075).