Recombinant human desmoglein-2 protein (DSG2-1601H, Creative Biomart, Shirley, NY) was reconstituted per manufacturer’s instructions and diluted to a concentration of 100 ng/ul with the addition of 1X protease inhibitor (Thermo Fischer Scientific Halt protease inhibitor). The reconstituted protein was then aliquoted and stored at – 80 °C in cryovials for further analysis.
Initial validation of the protein detection was performed using a rabbit monoclonal anti-desmoglein-2 antibodies (DSG2 antibody, Abcam, Waltham, MA) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam, Waltham, MA) as secondary antibodies. Following validation of the recombinant desmoglein-2 protein, optimization of protein detection was performed using various concentrations of primary and secondary antibodies and quantities of recombinant protein. Based on the results of these optimization blots, the protocol for western blot analysis of study samples was developed.
All study samples were analyzed using a standardized protocol in a single laboratory. Recombinant desmoglein-2 proteins (Bio-Rad, Hercules, CA) were first denatured and reduced by boiling in 1 × Laemmli buffer with 2-mercaptoethanol. A total of 375 ng of recombinant desmoglein-2 protein was loaded into each well of precast 4–20% polyacrylamide gels (Mini-protean TGX gels, BioRad, Hercules, CA), alternating with loading of a protein ladder (BioRad, Hercules, CA) and separated by SDS-PAGE before transferring to polyvinylidene difluoride membrane (Immun-blot, BioRad, Hercules, CA) at 60 V for 70 min. Adequate protein transfer was confirmed via Ponceau S solution (Sigma Aldrich, St. Louis, MO). Blots were then blocked with 10% bovine serum albumin (BSA) in Tris buffered saline with 0.05% Tween (TBST) (overnight, 4 °C). Blots were then cropped into sections so that each lane containing the recombinant protein was paired with a protein ladder. Canine sera were thawed at room temperature and diluted 1:100 in TBST with 5% BSA. The cut blots were then incubated in sera from a single study individual for two hours at room temperature, followed by washing in and secondary body incubation with rabbit anti-dog secondary antibody (1:20,000, Abcam, 136759) for one hour at room temperature. After washing, immunoreactive bands were detected using an enhanced chemiluminescence kit (Prometheus ProSignal Dura, Genesee Scientific, El Cajon, CA) and imaged using commercially available imaging system (ProteinSimple FluorChem E system) with an exposure of 60 s. Detected bands were then quantified via densitometry using the ImageJ software correcting for the background density of each blot by a single investigator in a blinded manner.
Initial validation of the protein detection was performed using a rabbit monoclonal anti-desmoglein-2 antibodies (DSG2 antibody, Abcam, Waltham, MA) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam, Waltham, MA) as secondary antibodies. Following validation of the recombinant desmoglein-2 protein, optimization of protein detection was performed using various concentrations of primary and secondary antibodies and quantities of recombinant protein. Based on the results of these optimization blots, the protocol for western blot analysis of study samples was developed.
All study samples were analyzed using a standardized protocol in a single laboratory. Recombinant desmoglein-2 proteins (Bio-Rad, Hercules, CA) were first denatured and reduced by boiling in 1 × Laemmli buffer with 2-mercaptoethanol. A total of 375 ng of recombinant desmoglein-2 protein was loaded into each well of precast 4–20% polyacrylamide gels (Mini-protean TGX gels, BioRad, Hercules, CA), alternating with loading of a protein ladder (BioRad, Hercules, CA) and separated by SDS-PAGE before transferring to polyvinylidene difluoride membrane (Immun-blot, BioRad, Hercules, CA) at 60 V for 70 min. Adequate protein transfer was confirmed via Ponceau S solution (Sigma Aldrich, St. Louis, MO). Blots were then blocked with 10% bovine serum albumin (BSA) in Tris buffered saline with 0.05% Tween (TBST) (overnight, 4 °C). Blots were then cropped into sections so that each lane containing the recombinant protein was paired with a protein ladder. Canine sera were thawed at room temperature and diluted 1:100 in TBST with 5% BSA. The cut blots were then incubated in sera from a single study individual for two hours at room temperature, followed by washing in and secondary body incubation with rabbit anti-dog secondary antibody (1:20,000, Abcam, 136759) for one hour at room temperature. After washing, immunoreactive bands were detected using an enhanced chemiluminescence kit (Prometheus ProSignal Dura, Genesee Scientific, El Cajon, CA) and imaged using commercially available imaging system (ProteinSimple FluorChem E system) with an exposure of 60 s. Detected bands were then quantified via densitometry using the ImageJ software correcting for the background density of each blot by a single investigator in a blinded manner.
Free full text: Click here
