Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Mutations of six amino acid residues in a B domain-deleted blood coagulation factor VIII have a cumulative effect on increasing its secretion

doi: 10.1016/j.rpth.2025.103325

Figure Lengend Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified WT and X6. The preparations containing 0.6 RUm, 1.2 RUm, 2.4 RUm, and 4.8 RUm of protein (from left to right respective lanes) were loaded on 4% to 12% (gradient) gel and subjected to electrophoresis followed by Coomassie blue staining. Single chain, heavy chain, light chain, A2-A3-C1-C2, and A1/A2 derived correspond to respective FVIII fragments (domains); von Willebrand factor is bovine protein from cell culture medium; S1, a B-domain-FVIII standard (moroctocog alfa); M, molecular weight markers. Multiplicity of heavy chain/light chain bands is related to structural variability (in glycosylation, etc.). The identity of protein bands was confirmed by using anti-FVIII polyclonal antibodies , MS, and a monoclonal anti-A2 antibody (GMA-012). FVIII, factor VIII; RUm, relative units of mass.

Article Snippet: Anti-Human FVIII:C Affinity Purified (Polyclonal) (sheep immunoglobulin [Ig]G) antibodies (CL20035AP) were from Cedarlane Laboratories and mAb GMA-012 (Mab HFVIII R8B12) targeting an epitope on FVIII, formed by residues 497-510 and 584-593, was from Green Mountain Antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Purification, Electrophoresis, Staining, Derivative Assay, Cell Culture, Molecular Weight, Glycoproteomics