Strepmab classic hrp

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Mini-PROTEAN^®Tetra System (Bio-Rad, Feldkirchen, Germany). InstantBlue (expedeon, Heidelberg, Germany) was applied for Coomassie stainings. For Western blot analysis a TRANS-BLOT ^® SD semi-dry transfer cell (Bio-Rad) was used. Stainings were performed with an Anti-StrepTag specific antibody (StrepMAB-Classic-HRP, IBA Lifesciences). Images were acquired with a ChemiDoc Imaging System (Bio-Rad).

Strain DS266 was grown in 500 mL MB medium for approximately 24 h at 30°C until reaching an OD578 of 0.8 either in the dark, under blue light (467 nm) or white light conditions, respectively. To crosslink the proteins in vivo formaldehyde to a final concentration of 0.125% (vol/vol) was added and the bacteria were further incubated for 20 min at 30°C under continuous shaking. Subsequently, free aldehyde groups were quenched by addition of 135 mM glycine and incubation was continued for 5 min at 30°C. Next, the bacteria were harvested by centrifugation (11,300 × g for 20 min), the pellet was washed in 50 mM Tris–HCl buffer (pH 8.2) and 500 mM NaCl and finally resuspended in 6 mL of the same buffer and 1 μL of benzonase (25 U/μL; Merck, Darmstadt, Germany) was added. For Strep-Tactin purification of the crosslinked Dshi_1135 protein complexes, cells were disrupted using a French Press (19,200 psi). The cell homogenates were clarified by centrifugation (121,000 × g for 60 min at 4°C) and the supernatant was subjected to Strep-Tactin column chromatography. Elution fractions were further analyzed by nanoLC-MS/MS, SDS-PAGE, and Western blotting. Western blots were probed with StrepMAB-Classic HRP (IBA Lifesciences, Göttingen, Germany) and polyclonal anti-Dshi_1135 antiserum (Davids, Regensburg, Germany).