Glu plasminogen

Human proteins used for ELISA include: plasma fibrinogen (Code: F3879), plasma fibronectin (Code: 11051407001), Glu-plasminogen (Code: P7999), vitronectin (Code: SRP3186), laminin (Code: L6274), and lactoferrin (Code: L1294) which were all supplied by Sigma.
Binding affinity measured by ELISA was performed as described previously^{17 (link)}. Recombinant protein RP15 was produced as described^{55 (link)} and both C-terminal peptides were synthesised by Chempeptide Limited (China). P1-30 (¹⁵⁹⁷TSAAKPGAPRPPVPPKPGAPKPPVQPPKKPA¹⁶²⁷) without any tags, but P1-15 (¹⁶¹³PGAPKPPVQPPKKPA¹⁶²⁷) was sequenced with an N-terminal biotin tag.
15 µg/ml of C-terminal P1 fragments were bound to wells and incubated with different host proteins. Wells were then incubated with different antiserum raised against the different host proteins at the following dilutions (all from Sigma): anti-fibrinogen 1:3000, anti-fibronectin 1:1000, anti-plasminogen: 1:2500, anti-vitronectin 1:5000, anti-laminin 1:750, and anti-lactoferrin 1:5,000. These incubations were followed by incubations with anti-rabbit IgG (Dako) or anti-goat IgG (both 1:2,000). Detection was measured by adding Tetramethylbenzidine (Sigma) followed by 1 M HCl, and absorbance was measured at 450 nm (620 nm as reference).

Glu-plasminogen, Lys-plasminogen, single-chain urokinase-type plasminogen activator (pro-uPA), trypsin inhibitor from Glycine max (SBTI), 6-aminohexanoic acid (EACA), tranexamic acid (TXA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Shanghai, China). The chromogenic substrate S-2444 for urokinase-type plasminogen activator (uPA) was purchased from BioMed (Shanghai, China). Absorbance was measured using a microplate reader (SH-1000 Lab, Corona Electric, Ibaraki, Japan). Fungi fibrinolytic compound 1 (FGFC1) was extracted and purified in our laboratory (purity > 98%). All other chemicals were of analytical grade.