Spectra multicolor low range protein ladder

Approximately 20 μg of purified Babesia sp. BQ1 (Lintan) recombinant proteins (rRAP-1a61-1 and rRAP-1a61-1/CT) were separated using SDS-PAGE in 18 % polyacrylamide gels. The proteins were transferred to nitrocellulose (NC) membranes with a 0.45 μm pore size (BioRad) at 24 V and 50 W for 35 min. The membrane was then cut into 0.25 cm strips and incubated in a blocking solution [5 % skimmed milk powder in Tris-buffered saline (pH 7.6) with 0.1 % Tween-20 (TBST)] for approximately 3 h at 4 °C on a shaker. After the NC strips had been washed three times, they were incubated for 1 h with each tested serum (diluted at 1:100 in TBST). After the strips were washed three times in TBST, they were incubated for 2 h with monoclonal anti-goat/sheep IgG-alkaline phosphatase conjugates (Sigma, A8062, dilution: 1:5000). The strips were then washed three times with TBST and incubated in a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) liquid substrate system (B1911-100ML, Sigma) for 15 to 20 min. The approximate molecular weights of the revealed proteins were evaluated by comparing their migration to a standard molecular weight marker (SM0671, 10–170 kDa, PageRuler Prestained Protein Ladder; SM1861, 1.7–40 kDa, Spectra Multicolor Low Range Protein Ladder, Thermo Scientific).

Bacteria were cultured to A₆₀₀ ~0.5. One milliltre was harvested by centrifugation, washed with PBS, then repelleted and resuspended in a volume of 1× Novex Tricine SDS sample buffer (Invitrogen) normalized for cell density (50 µl per 1 ml A₆₀₀ 0.5). Samples were boiled for 15 min and either cooled on ice (no proteinase K) or incubated with proteinase K (NEB) at 55 °C for 1 h. Samples were re-boiled and electrophoresed using the tricine buffer system with Novex tricine 16%-acrylamide gels (Invitrogen). Spectra Multicolor Low Range Protein Ladder (Thermo) was included to indicate approximate molecular weights. Gels were fixed, washed, stained using Pro-Q Emerald 300 (Invitrogen), and imaged using UV transillumination (Biorad Chemidoc MP). Gels were subsequently stained with Coomassie Brilliant Blue for detection of total protein. Image lab software (Biorad) was used to quantify LOS or total protein intensity levels. Samples were normalized by dividing the LOS intensity level of each band region by the total protein level from Coomassie staining. Relative values were calculated by dividing each normalized LOS value by the total normalized LOS levels in WT.

For direct protein extraction, the samples were mixed with the denaturing and reducing sample buffer (125 mmol/L Tris-HCl pH 6.8, 4% SDS, 20% v/v glycerol, DTT 50 mg/mL), boiled at 100 °C for 5 min, and centrifuged. Then, SDS-PAGE electrophoresis was performed using the method of Schägger–von Jagow [25 (link)]. The PageRuler Prestained Protein Ladder and Spectra Multicolor Low Range Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) were used as molecular weight protein markers. Gels were run in the Mini PROTEAN Tetra Cell electrophoresis equipment (Bio-Rad Laboratories, Hercules, CA, USA) and stained with Coomassie Brilliant Blue R-250.