Acetate buffer

Biotinylated heparin was synthesized by adapting a previously reported procedure (Thakar et al., 2014 (link)). First, a solution containing heparin (4 mM, Sigma-Aldrich), 10 mM acetate buffer (made from glacial acetic acid (Carl Roth, Karlsruhe, Germany) and sodium acetate (Sigma-Aldrich) at pH 4.5) and aniline (100 mM, Sigma-Aldrich) was prepared. Biotin-PEG₃-oxyamine (3.4 mM, Conju-Probe, San Diego, United States) was added to the heparin solution and allowed to react for 48 h at 37°C. The final product was dialyzed against water for 48 h by using a dialysis membrane with a 3.5 kDa cutoff. The final solution was then lyophilized and stored at −20°C. For further use, the conjugates were diluted to the desired concentrations in buffer. The obtained biotinylated heparin was characterized by biotin-streptavidin binding assays using QCM-D. The average mass of the heparin isolate, when anchored to the surface, was estimated at 9 kDa (∼18 disaccharide units) by QCM-D analysis (Srimasorn et al., 2022 (link)).

Biotinylated heparin was synthesized by adapting a previously reported procedure⁸¹ . A solution containing heparin (4 mM, Sigma-Aldrich) in 100 mM acetate buffer (made from glacial acetic acid (Carl Roth, Karlsruhe, Germany) and sodium acetate (Sigma-Aldrich) at pH 4.5) containing aniline (100 mM, Sigma-Aldrich) was prepared. Biotin-PEG₃-oxyamine (3.4 mM, Conju-Probe, San Diego, USA) was added to the heparin solution and allowed to react for 48 h at 37 °C. The final product was dialyzed against water for 48 h by using a dialysis membrane with a 3.5 kDa cutoff. The final solution was then lyophilized and stored at −20 °C. For further use, the conjugates were diluted to the desired concentrations in the buffer. The obtained biotinylated heparin was characterized by biotin-streptavidin binding assays using QCM-D. Low-sulfated HS was derived from porcine intestinal mucosa (Celsus Laboratories, Cincinnati, OH, USA), and high-sulfated HS was purified in the laboratory of Hughes Lortat-Jacob (Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France) and treated in the same manner. The average mass of all the heparin and HS isolates, when anchored to the surface, was estimated at 9 kDa (~18 disaccharide units) by QCM-D analysis^{82 (link)}.