Based on the ELISA data, the calculated K D for the recombinant proteinLsa33 with PLG is 23.53 ± 4.66 nM (Figure 6C). This K D

value is in the same order of magnitude with the ones obtained with several recombinant proteins in our laboratory [21]. Figure 6 Recombinant proteins PD0332991 cost binding to serum components. (A) Human purified PLG, factor H and C4bp (10 μg/ml) were coated onto ELISA plates and allowed to interact with the recombinant proteins Lsa33 and Lsa25 (10 μg/ml). Gelatin and fetuin were used as negative controls for nonspecific binding. The binding was detected by antibodies raised against each recombinant protein (1:750). Bars represent the mean of absorbance at 492 nm ± the standard deviation of three replicates for each protein and are representative of three independent experiments. For statistical analyses, the binding of Lsa33 and Lsa25 was compared to its binding to gelatin by two – tailed t test (*P < 0.05 and **P < 0.005). (B) Similar as described in (A) but the binding of the recombinant proteins was detected by anti - polyhistidine monoclonal antibodies (1:200). Included

LY2109761 chemical structure is a His – tag recombinant protein Lsa63 that does not bind C4bp. (C) Recombinant proteins dose – dependent binding experiments with PLG. The binding was detected by polyclonal antibodies against each protein; each point was performed in triplicate and expressed as the mean absorbance value at 492 nm ± standard error for each point. Gelatin was included as a negative control. The dissociation

constant (KD) is depicted and was calculated based on ELISA data for the recombinant protein that reached equilibrium. (D) Plasmin generation by PLG bound to recombinant proteins was assayed by modified ELISA as immobilized proteins received the following treatment: PLG + uPA + specific plasmin substrate (PLG + uPA + S), or controls lacking one of the three components (PLG + uPA; PLG + S; uPA + S). Lsa63 and BSA were employed as negative controls. Bars represent mean absorbance at 405 nm, as a measure of relative substrate degradation ± the standard deviation of four replicates for Selleck Forskolin each experimental group and are representative of three independent experiments. Statistically significant binding in comparison to the negative control (BSA) are shown: *P < 0.05. (E) Recombinant proteins dose – dependent binding experiments with C4bp. The binding was detected by polyclonal antibodies raised against each protein (1:750); each point was performed in triplicate and expressed as the mean absorbance value at 492 nm ± standard error for each point. Gelatin was included as a negative control.