G box chemi xx6 imager

Lactobacillus strains were streaked onto MRS agar and grown for 48 h under anaerobic conditions at 37 °C. Colonies from each strain were picked and then patched onto a fresh MRS agar plate fitted with a 50-square grid. After 48 h, an 82-mm nitrocellulose membrane (Whatman Protran BA85) was laid onto the plates, then lifted, along with adherent bacterial growth and placed into PBST buffer (1× PBS with 0.05% Tween-20, pH 7.4). Blots were washed 3× in PBST at room temperature (RT). The blot was blocked in PBST + 5% BSA and washed 3× in PBST. Then they were incubated with 2 µg/ml purified nanobody proteins in PBST + 5% BSA for 1 h as described above. Finally, blots were washed in PBST and then incubated with a 1:5000 dilution of HisProbe HRP-conjugated antibody (Thermo Scientific) for 1 h at RT with gentle shaking (50 RPM). After final washing, blots were incubated with ECL Plus per manufacturer’s instructions (Cytiva). For the fluorescent nanobody assay, the blots were incubated with Lc58-RFP (50 µg/ml) and Lj75GFP (25 µg/ml), washed, and then imaged. The blots were imaged with a Gbox Chemi XX6 Imager (Syngene) using the ECL western blot program for non-fluorescent nanobody binding and Texas Red and TurboGFP filters to detect fluorescent nanobody binding. Image analysis was conducted with GeneTools Software.

Protein was extracted from 1 × 10⁶ cells pre-treated with varying concentrations of the B. carterii oleoresin methanolic extract for 48 h using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, United States). Protein was quantified using bicinchoninic acid protein assays. A concentration of 50 µg of whole cell protein lysate for each condition was separated using sodium dodecyl sulphate (SDS) PAGE over a gradient of 4%–20% (Merck, United States) and transferred to 0.45 µm Polyvinylidene fluoride (PVDF) membranes (Immobilon, United States) as described in Mahmood and Yang (2012) (link). The primary and secondary antibodies utilised are described within the Supplementary Section S1.2. Protein expression was then visualised by chemiluminescence using Crescendo Western horseradish peroxidase (HRP) substrate (Immobilon, United States). Chemiluminescent Images were taken using a G:Box Chemi-XX6 imager and the GeneSys V1.5.2.0 software (both Syngene, United States). Protein abundance was quantified using the software package ImageJ (National Institutes of Health, United States).

Competitive ABPP on crude lysates from ∼10⁷ saponin-isolated parasites or uninfected erythrocytes was performed as described above. Following in-gel fluorescence scanning, proteins were transferred to a nitrocellulose membrane and TAMRA fluorescence was imaged with a G:Box ChemiXX6 imager (Syngene, Frederick, MD). The membrane was then blocked with 2% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST/BSA) for 1 h at room temperature, incubated with primary antibody diluted in TBST/BSA for 1 h, and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000; GE Healthcare Life Sciences, Piscataway, NJ) for 1 h. The primary antibody used was affinity-purified anti-APEH rabbit polyclonal antibody (IgG) raised against an APEH fragment consisting of amino acids 381 to 732 (product number 14758-1-AP; Proteintech, Rosemont, IL; 0.26 µg/ml). Chemiluminescent signal was developed with ECL Plus (GE Healthcare life Sciences, Piscataway, NJ) and detected using a G:Box ChemiXX6 imager. Image alignment (using molecular markers) and contrast adjustment were accomplished with Adobe Photoshop CS2 (Adobe, Inc., San Jose, CA).