CHAPTER 5
Donkey Anti-Sheep IgG, pcDNA6/V5-His A, Gibco Penicillin-Streptomycin, EZ-Link SulfoNHS-LC-Biotin, Lipofectamine 2000 and One-shot Bl21 pLysS Escherichia coli (E.coli) were from Life Technologies (Carlsbad, CA, USA). Polyclonal antibodies (total IgG) to FXII, PPK, and C1inh (affinity purified) were from Cedarlane Ltd. (Burlington, ON, Canada). Polyclonal affinity purified antibodies to Plasminogen and HK were from Affinity Biologicals Inc (Ancaster, ON, Canada). Peroxidase-conjugated polyclonal rabbit anti-sheep antibody was from Dako (Heverlee, Belgium). Blasticidin-S hydrochloride and bradykinin ELISA were from Enzo Life Sciences (Raamsdonksveer, the Netherlands). Sodium acetate and CaCl2 were from Merck (Darmstadt, Germany). Immobilon-FL, Ficoll 400, and ethanol were from Merck-Millipore (Amsterdam, the Netherlands). Chromogenic substrates H-DVal-Leu-Lys-AMC (I1390) and H-D-Pro-Phe-Arg-pNA (L2120) were from Bachem (Bubendorf, Switzerland). FXII immune-depleted plasma and Phe-Pro-Argchloromethylketone (PPACK) were purchased from Haematologic Technologies Inc. (Essex Junction, VT, USA). α-FXIIa, β-FXIIa and active plasma kallikrein were purchased from Enzyme Research laboratories (ERL; South Bend, IN, USA). Pfu Turbo was from Agilent Technologies (Santa Clara, CA, USA) and T4 ligase from New England Biolabs (Whitby, ON, Canada). HEK293T cells were from ATCC (LGC Standards GmbH, Wesel, Germany) and fetal bovine serum (FBS) was from Lonza (Breda, the Netherlands). Streptactin superflow sepharose was from IBA-life sciences (Goettingen, Germany). Lysinesepharose and HiLoad 26/600 Superdex 200 pg columns were from GE Healthcare (Hoevelaken, the Netherlands). Tris-HCl was from Roche (Woerden, the Netherlands) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from VWR International (Amsterdam, the Netherlands). Kaolin (light) was purchased from BDH ltd (Poole, UK) and routinely suspended in in Tris-buffered Saline (TBS; 50 mM Tris-HCL, 150 mM NaCl, pH=7.4). Odyssey blocking reagent was purchased from Licor (Bad Homburg, Germany) and Streptokinase (Streptase) from CSL-Behring (Breda, the Netherlands). 3,3’,5,5’-Tetramethyl-benzidine (TMB) was obtained from Tebu Bio (Heerhugowaard, the Netherlands). Kininase inhibitors Apstatin, Ramipril, Lisinopril, and Enalapril were purchased from Santa Cruz biotechnology Inc. (Santa Cruz, CA, USA). C1-esterase inhibitor was from Alpha diagnostics (San Antonio, TX, USA) and Streptavidin-poly-HRP from Sanquin Blood Supply (Division Reagents, Amsterdam, the Netherlands). Technozym PAP Complex ELISA Kit was obtained from Technoclone (Vienna, Austria). STACHROM antiplasmin activity assay kit was purchased from Diagnostica Stago S.A.S (Asnières sur Seine, France). In silica prediction Mature amino acid sequences (Uniprot: P00748219) were analyzed with Peptide Cutter software for the presence of putative cleavage sites that are sensitive to trypsin-like enzymes220. Changes in protein charge were analyzed with Protparam220 and changes in O-linked glycosylation with NetOGlyc server 4.0221.
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