Cta fplc system

In order to analyze the structural properties of NfCPI, electrophoretic analysis of the NfCPI was performed in the presence and absence of SDS or β-mercaptoethanol [β-ME, 5% (vol/vol)] with or without prior heating at 100 °C for 5 min [12 (link),13 (link)]. The native molecular size of NfCPI was also analyzed by gel filtration chromatography using a Superdex 200 HR 10/30 column with an Äcta FPLC system (GE Biosciences, Pittsburgh, PA, USA). The purified NfCPI (1 mg) was loaded onto the column, and the collected fractions (0.5 mL) were analyzed by SDS-PAGE, followed by measurement of their inhibitory activities against NfCB and NfCBL. The column was calibrated with gel filtration size marker proteins (Sigma): blue dextran (2000 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). The K_av value of each size marker protein was calculated using the equation K_av = (V_e − V₀)/(V_t − V₀), where V_e is the elution volume of protein_,V₀ refers to the elution volume of blue dextran, and V_t is the total bed volume.

To analyze the native molecular size and structure of fowlerstefin, gel filtration chromatography was performed with a Superdex 200 HR 10/30 column using an Äcta FPLC system (GE Biosciences, Pittsburgh, PA, USA). Purified recombinant fowlerstefin (1 mg) was loaded onto the column and fractions (0.5 ml each) were collected. The collected fractions were separated via SDS-PAGE and their inhibitory activities against NfCPB-L were determined. The column was calibrated with the following molecular weight markers (Sigma-Aldrich): blue dextran (2000 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). The K_av value of each protein was calculated based on the equation K_av = (V_e − V₀)/(V_t − V₀), where V_e is the elution volume of the protein, V₀ denotes the elution volume of blue dextran and V_t refers to the total bed volume. The molecular structure of fowlerstefin was further analyzed using electrophoretic methods. Purified recombinant fowlerstefin (20 μg) was electrophoresed in the presence and absence of SDS without heating as described previously [25 (link)]. The samples treated as above were analyzed via SDS-PAGE followed by Coomassie blue staining.