Sds containing sample buffer

1 vial of Helixate FS (Bayer Healthcare) containing 252 IU was diluted with 2 ml water (final concentration = 0.126 IU/μl containing 12.6 ng/μl of rFVIII protein). For gel separation, 22 μl of 12.6 ng/μl rFVIII was diluted with 10 μl of SDS-containing sample buffer (BioRad) and heated at 95°C for 5 min. 15 μl of this mixture was loaded per gel lane containing ~130 ng of rFVIII.

Tumor lysates were prepared in RIPA buffer (ThermoFisher) containing a 10% protease inhibitor cocktail (ThermoFisher) using a gentleMACS device (Miltenyi Biotech) based on the manufacture’s protocol. For ELISA detection, anti-hinge rabbit polyclonal antibodies (2 μg/ml) were coated on high-binding 96-well plates to capture scIgGs in tumor extracts; and anti-mouse specific antibody at 1:5000 dilution (Jackson Immune Research Laboratory, PA) with HRP conjugation was used for detection. For Western blotting detection, protein A magnetic beads were incubated with cell lysates at 4 °C for 1 hour to capture total IgGs including both intact IgGs and scIgGs, and the beads were collected and incubated with SDS containing sample buffer (Bio-Rad) at 95 °C for 5 min. The eluted samples were subjected to SDS-PAGE separation and WB detection using a goat anti-mouse IgG-Fc-HRP conjugate (1:4000) (Jackson Immune Research Laboratory, PA) as previously described^{10 (link),12 (link)}.