Mini trans blot cell

Purified GlpQ (500 ng) was electrophoresed on each replicate lane of a precast mini 4%�?"20% sodium dodecyl sulfate�?"polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a nitrocellulose membrane using the Bio-Rad MiniTrans Blot Cell (Bio-Rad Laboratories). Replicate strips containing rGlpQ were blocked overnight at 4A�C in PBS (pH 7.2)/5% dried milk/0.05% Tween 20. The blocked strips were then individually incubated with human serum at a 1:250 dilution at room temperature in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20 for 1 h. The strips were then washed 3 times and incubated for 1 h with horseradish peroxidase�?"conjugated rabbit anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA) or with horseradish peroxidase�?"conjugated goat anti-human IgM (Invitrogen) at a 1:5,000 dilution in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20. Bound antibodies were detected by using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Serum from �%^10% of the study participants reacted to a �%^55-kDa band, presumably a trace contaminant copurified with the rGlpQ generated in a bacterial expression system. Samples with a 39-kDa band corresponding to GlpQ on positive control mouse serum samples were considered GlpQ antibody�?"positive (Figure 1).

To monitor the purification process and confirm rgD5 antigenicity prior to the ELISA development, SDS-PAGE was carried out on 12% polyacrylamide gel. The gels were either stained overnight with Coomassie Blue R-250 (Bio-Rad) or electroblotted onto nitrocellulose membrane (Bio-Rad) using Bio-Rad Mini Trans-Blot Cell (Bio-Rad). Briefly, the membrane was blocked with 5% non-fat milk and, after three washes in phosphate buffer saline containing 0.05% Tween-20 (PBS-T) (137 mM NaCl, 2.7 mM KCl, 100 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4), the membrane was incubated with mouse monoclonal antibody (MAb) anti-6xHis HRP conjugated (Invitrogen) or with polyclonal antibody (PAb) anti-BoHV-5 from bovine experimentally immunized with inactivated BoHV-5 as previously described [22 (link)]. The immunoblot was developed using Sigma FAST 3,3’-Diaminobenzidine (DAB) with Metal Enhancer tablets (Sigma). The protein concentration was determined by bicinchoninic acid (BCA) protein assay (Pierce) method according with the manufacturer’s instructions.

Total cellular lysates were generated with RIPA buffer (Sigma) containing protease inhibitors (Roche). Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific). Equal protein quantities were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore) using a Mini Trans-Blot Cell (Bio-Rad). Blots were probed with the relevant antibodies, and proteins were detected using enhanced chemiluminescence reagent (Thermo Scientific).

Protein bands were run in a 15% polyacrylamide gel and transferred to a PVDF membrane using a mini trans-blot cell (Bio-Rad, USA). The membrane was stained with Coomassie blue R-250 and the bands were cut with a sterilized scalpel. Protein bands were identified using the Edman reaction and a Procise 491 HT Protein Sequencer to determine the N-terminal sequences (Analytical Laboratory, Analytical Services Unit, ITQB NOVA).

Cells were harvested with NP-40 lysis buffer (ThermoFisher Scientific, UK) with protease and phosphatase inhibitors cocktails (Sigma‐Aldrich). Samples were loaded on 7.5% or 4–20% gels and resolved by electrophoresis. Proteins were transferred to PVDF membranes using Mini Trans-Blot Cell (BioRad). Membranes incubated for 1 h in blocking solution consisting of 5% w/v milk diluted in TBS-T 145 mM NaCl, 20 mM Tris-base, pH 7.4, 0.5% Tween-20 and labelled with primary antibody overnight at 4 °C for DHC (Proteintech—1:1000), Rab46 (CRACR2A: Proteintech 1:800), p150^Glued (BD Biosciences—1:1000), ATP1α1 (Santa Cruz—1:500), GFP (ThermoFisher—1:1000) and histidine (BioRad—1:1000). Immunoblots visualised using HRP-conjugated donkey anti-mouse, anti-rabbit secondary antibodies (Jackson ImmunoResearch—1:10,000) and SuperSignal Femto (Pierce).