Anti Flagellin  

Western blot showing detection of 0.1 μg of recombinant Flagellin protein. Lane 1: Molecular weight markers. Lane 2: MBP-Flagellin fusion protein (arrowhead at expected MW: 76.3 kDa). Lane 3: MBP alone. Protein was run on a 4-20% gel, then transferred to 0.45 μm nitrocellulose. After blocking with 1% BSA-TTBS (p/n MB-013, diluted to 1X) overnight at 4°C, primary antibody was used at 1:1000 at room temperature for 30 min. HRP-conjugated Goat-Anti-Rabbit (p/n 611-103-122) secondary antibody was used at 1:40,000 in MB-070 blocking buffer and imaged on the VersaDoc^TM MP 4000 imaging system (Bio-Rad).

Western Blot of Anti-Flagellin Antibody. Lane 1: Flagellin Control Protein (p/n 000-001-C14) Reduced [0.1μg]. Lane 2: Opal Prestained Molecular Weight Markers (p/n MB-210-0500). Lane 3: Flagellin Control Protein Non-Reduced [0.1μg]. Primary Antibody: Anti-Flagellin at 1.0μg/mL overnight at 4°C. Secondary Antibody: Goat anti-Rabbit IgG peroxidase (p/n 611-103-122) at 1:70,000 for 30 mins at RT. Block: BLockOut (p/n MB-073) for 1hrs at RT. Predicted MW: ~76.3kDa. Exposure: 2sec.

(B) A1arp+/vlsE+？spirochetes were probed with anti-FlaB antibodies (αFlaB) to verify outer membrane integrity during immunofluorescence. The anti-FlaB antibodies could access the periplasmic flagellar protein only when permeabilized with methanol first (bottom row). Each immunofluorescence assay was conducted three times with two independent sets of clones. The scale bar represents 50 μm. meth. fix., methanol fixation. Fig 2.PMID:？32187539

BB0405 is expressed on the surface of？B. burgdorferi？sensu stricto and？B. afzelii. Viable spirochetes were incubated with (+) or without (−) proteinase K for 30？min and 60？min and processed for immunoblot analyses with antibodies against BB0405. Flagellin B antibodies served as a subsurface control. Fig 3. PMID:？33637813

HS-198 accumulation and localization in？B. burgdorferi. (A) Fluorescent imaging of？B. burgdorferi？in spirochetes, blebs, and aggregates stained with HS-198 (HtpG), WGA-488 (extracellular matrix), and DAPI (DNA). Images shown are representative of？n？=？48. (B) Confocal images (100×) of HS-198 and flagellin antibody staining of a spirochete (n？=？5). (C) Correlative light electron microscopy (CLEM) of HS-198 fluorescence overlaying an EM image of a？B. burgdorferi？spirochete. Outer membrane (OM), protoplasmic cylinder (PC), and periplasmic space (PS) are identified. Images shown are representative of？n？=？4 spirochetes with TEM sections of 65？nm. Fig 2. PMID:？33910962