Rabbit anti c4bpa antibody

Gc (10^8) was pelleted, washed, and incubated with 50 μg/mL C4BP diluted in PBS+ for 20 minutes at 37°C. Gc were pelleted, washed, and resuspended in 5 μg/mL rabbit anti-C4BPA antibody (Novus Biologicals 88262), diluted in RPMI + 10% FBS, for 30 minutes at 37°C. Gc were pelleted and resuspended in 5 μg/mL goat anti-mouse AF488 (Invitrogen, Carlsbad, CA) diluted in RPMI + 10% FBS for 30 minutes at 37°C. The samples were fixed in 2% PFA with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for visualization of the bacteria. Samples were analyzed by imaging flow cytometry using Imagestream^X Mk II with INSPIRE software (Luminex Corporation) at the Flow Cytometry Core Facility at the University of Virginia, and data were analyzed using IDEAS software.

Proteins or Gc were boiled for 5 minutes in reducing sample buffer containing 60 mM Tris pH 6.8, 25% glycerol, 0.4% SDS, 5% β-mercaptoethanol, and 0.1% bromophenol blue. Proteins or lysates were resolved on a 4–20% gradient SDS-polyacrylamide gel (Criterion TGX 5671094, Bio-Rad), transferred in Towbin [62 (link)] buffer to nitrocellulose membrane, and the membrane was incubated in 5% BSA in Tris buffered saline with 0.1% Tween for 1 hour (blocking buffer). C4BP was detected using rabbit anti-C4BPA antibody (Novus Biologicals 88262) followed by goat anti-Rabbit H+L Alexa Fluor 680 cross adsorbed secondary antibody (Invitrogen A21109), and Opa or porin (loading controls) were detected with mouse anti-Opa 4B12 and mouse anti-porin H5.2 (gift from Marcia Hobbs) respectively followed by goat anti-Mouse IgG (H+L) Dylight 800 cross adsorbed secondary antibody (Invitrogen SA5-10176). All antibodies were diluted in blocking buffer. Bands were visualized via LI-COR Odyssey.