Transferrin binding protein B and Transferrin binding protein A 2 expand the transferrin recognition range of Histophilus somni

Anastassia K. Pogoutse; Trevor F. Moraes

shaking. Cells were harvested by centrifugation for 10 minutes at 4000g, washed once in PBS 380 containing 10 mM MgCl 2 , and fixed for 20 minutes in 1.85% formaldehyde solution (3.7% 381 formaldehyde mixed with equal parts PBS + 10 mM MgCl 2 ). Cells were washed once in PBS + 382 10 mM MgCl 2 and resuspended at an OD 600 of 10 in the same buffer. 100 uL of cells per well 383 was applied to 96-well flat-bottomed tissue culture plates (Sarstedt). Plates were incubated for 3 384 hours at room temperature in a biosafety cabinet. Plates were then washed once with PBS and 385 patted dry. 50 uL of 6.6 nM biotinylated transferrin and 50 uL of 132 nM of unlabeled transferrin 386 was applied to each well and left to incubate for 2 hours at room temperature. Transferrin was 387 diluted in 5% skim milk dissolved in PBS. Plates were washed 3 times with PBS and 100 uL 388 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/730739 doi: bioRxiv preprint volumes of streptavidin-HRP at a dilution of 1 in 5000 dissolved in skim milk solution was 389 added to the wells and left to incubate for one hour at room temperature. Plates were washed 3 390 times with PBS. Binding was detected with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate 391 (Sigma), reactions were stopped with 2 M HCl, and absorbance was read at 450 nm. Signal of 392 wells containing buffer instead of cells was subtracted from all reads. Wells including cells but 393 not labeled transferrins were also used to monitor for nonspecific binding. Absorbance readings 394 were normalized to the signal of wells containing no competitor. Data was plotted and analyzed 395 in Graphpad Prism. Statistical analysis was performed using one-way ANOVA followed by 396