Chemidoc xrs system image lab software

Protein concentration from stool and brain samples was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Thirty µg of protein was loaded and separated on a 10–12% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Sigma). The membranes were blocked with 3% BSA in TBS Tween for 60 min at room temperature and incubated overnight at 4 °C with the primary antibody CsgA_ECO57 (major curlin subunit from E. coli) (Cusabio Technology LLC, Houston TX, USA.) at 1:3500. This antibody was previously validated [15 (link)]. After that, the membranes were incubated with the secondary antibody in the blocking buffer and blots were incubated with anti-rabbit (Abcam, AB6721) secondary antibody conjugated with horseradish peroxidase (1:20,000). GAPDH (1:40,000, ab181602) was used to normalize the data. Images were analyzed with ChemiDoc™ XRS + System Image Lab™ software (Bio-Rad, Hercules, CA, USA). The assays were performed three times using independent blots.

Whole cell lysates were prepared using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, 0.1% SDS and protease inhibitors). Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL) determined protein concentrations. Thirty µg of whole cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blotted with ARID3B antibody (Bethyl Laboratories, Montgomery, TX: Cat# A302-564A), and Histone H3 (D1H2) antibody (Cell Signaling, Danvers, MA) for loading control. Densitometry analysis was performed on three blots using BIORAD Chemidoc XRS+ System Image Lab Software (Hercules, CA).

The total protein of colon tissue samples of 4 patients per group (active and remission UC and control group) was extracted with solution of RIPA buffer (Sigma-Aldrich) and cocktail of protease inhibitor (Roche®), quantified by Bradford assay (Bio-Rad, Hercules, CA, USA), and stored at -70C. The protein detection was performed by electrophoresis in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. All blots were blocked with 3% BSA for 60 min at room temperature and incubated overnight at 4 with primary antibodies. The primary antibodies for CFTR/ABCC7 were used in a concentration of 1 : 1000, and β-actin was used to normalize the data in a concentration of 1 : 10000. The blots were incubated with anti-rabbit secondary antibodies conjugated with horseradish peroxidase (1 : 3000). Images were analyzed with the ChemiDoc™ XRS+ System Image Lab™ Software (Bio-Rad, Hercules, CA, USA), and the western blot analysis was performed using independent blots.