Ab19171

Western blot analysis was performed for assessment of plasma CP levels. Three Western blots were prepared containing plasma samples from neonates of the control group along with samples from infected neonates. An additional Western blot was prepared with samples from the group of infected neonates that were pre-selected with respect to the CRP levels. Plasma was diluted in ultrapure H₂O (Biochrom AG, Berlin, Germany) and 4x sample buffer (200 mM Tris-HCl, pH 7.5, 50% glycerin, 4% SDS, 0.04% bromophenol blue, and 125 mM DTT). Plasma proteins were size-fractionated by sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose membranes by semi-dry blotting (Optitran, Schleicher & Schuell, Dassel, Germany). Antibodies against CP (1:2000 dilution, ab19171, Abcam, Cambridge, UK) were used and bands visualized by chemiluminescence (Western-Bright Substrate Sirius, Biozym Scientific, Oldendorf, Germany) using the Fluor Chem FC2 detection system (Biozym Scientific). Quantification of Western blot signals was achieved by using the Java-based image processing program ImageJ (NIH, Bethesda, MD, USA).

All reagents used were from Sigma-Aldrich Co., MO, USA or Cayman Chemicals, MI, USA. Specific antibodies for each protein were used for Western blot (WB) analysis: rabbit polyclonal anti-soluble VCAM-1 (sc1504-R), goat polyclonal anti-4-hydroxynonenal-protein adducts (4-HNE) (sc-130083), and mouse monoclonal anti-β-actin were purchased from Santa-Cruz Biotechnology, TX, USA (sc47778). The mouse monoclonal anti-paraoxonase 1 (PON1) (ab24261), rabbit polyclonal anti-malondialdehyde-protein (ab27642), goat polyclonal anti-ceruloplasmin (ab19171), rabbit polyclonal anti-VCAM-1 (ab106777), rabbit polyclonal anti-MCP-1 (ab9669), mouse monoclonal anti-CRP (ab50861), rabbit polyclonal anti-ADAM17 (ab39162), and the secondary antibodies rabbit polyclonal anti-goat (ab6741-1), goat polyclonal anti-rabbit (ab6721), and rabbit anti-mouse (ab6728) labeled with horseradish peroxidase (HRP) were from Abcam, Cambridge, UK. The nitrocellulose membrane (0.4 μm or 0.22 μm) for WB was from BioRad Laboratories, Hercules, CA, USA. The Chemiluminescent HRP substrate (Immobilon Western) kit was from Millipore Corporation, Billerica, MA, USA.

Total protein content was quantified by Bradford method using bovine serum albumin (Pierce) as a standard, and human albumin 20% (Kedrion) as internal control. SDS-PAGE was performed using NuPAGE Novex 4-12% Bis-Tris acrylamide gel, MOPS SDS running buffer, NuPAGE LDS Sample buffer, and proteins were visualized by Simply Blue Safe Stain (ThermoFisher Scientific). CP purification intermediates were analyzed in semi-native denaturing (without boiling), denaturing (boiling samples at 90 °C for 10 min) and reducing conditions (by addition of NuPAGE antioxidant and Sample Reducing Agent, ThermoFisher Scientific). Cryocheck pooled normal plasma (Coachrom Diagnostica), together with commercially available research grade purified CP (ALX-200-089 ENZO LIFE SCIENCES) were used as reference. For Western blot (WB) proteins were transferred onto a nitrocellulose membrane with a Trans-Blot Turbo system (BioRad). Anti-CP antibody (ab19171, Abcam) and secondary antibody (Rabbit anti-goat Ig-HRP, Dako, Agilent P044901-2) incubations were carried out for 1 h at room temperature. Signals were revealed using chemiluminescence substrate (Clarity Western ECL Substrate – BioRad) on Chemidoc XRS+ (Bio-Rad Laboratories).