Ettan dige imager

For peptide ELISPOT the following peptides were designed:
peptide #1 - RVLAPALDSWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #2 - LPKFSAPSASGPGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #3 - ESTRYQLWLPHQGGGDYKDDD{LYS(BIOTIN)} (Genescript)
control peptide - AVLAAALASWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
In brief, 110 pg biotin-conjugated peptides were printed on nitrocelluose coated slides (10485323, Whatman) by SpotBot® 4 (Arrayit). For primary antibody human precleared serum (1:100) was used, for secondary antibody rabbit anti-human IgG (H&L) (HRP) (Abcam) was used. All incubations were done for 1 h at room temperature. Results were scanned using Ettan DigeImager (GE Healthcare Life Sciences) and images calculated using ImageQuant software version 8.1 (GE Healthcare Life Sciences).

Osteosarcoma and soft callus tissues were lysed in radioimmunoprecipitation assay buffer supplemented with 1% protease inhibitor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), homogenized and incubated on ice for 30 min. The supernatant was collected by centrifugation at 15,000 × g at 4°C for 20 min. Crude tissue lysates were further prepared for 2DE analysis using a 2-D Clean-Up kit, according to the manufacturer's protocol (GE Healthcare Life Sciences, Uppsala, Sweden). Protein concentration was determined using Bradford assay. The protein pellets were resuspended in 2D lysis buffer. Individual samples (60 µg protein) were applied by overnight in-gel rehydration of 7 cm pH 3-10 nonlinear gradient IPG strips (GE Healthcare, Chicago, IL, USA). IEF was performed at 7,000 Vh, 55 mA per gel strip using an Ettan IPGphor 3 (GE Healthcare). The IPG strips were equilibrated and separated in 14% SDS-PAGE followed by SYPRO Ruby staining (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described (16 (link)). Gels were scanned and visualized using Ettan DIGE Imager (GE Healthcare).

M13 phage particles displaying the relevant peptides were printed onto nitrocellulose coated glass slides (10,485,323, Whatman) using SpotBot® 4 arrayer (Arrayit) in four dilutions (1:2, 1:10, 1:100, 1:1000). 1xPBS-20% glycerol and mQ were used as negative controls and human IgG serial dilutions (12–50 ng/µl) as positive controls. Human serum or plasma (1:50), previously precleared to plastic and E. coli/wild type M13 phage antigens, was used as primary antibody while rabbit anti-human IgG H&L (HRP) (#ab6759, Abcam, RRID: AB_955,434) (1:1000) was used as a secondary antibody. Both antibody incubations were conducted at room temperature for 1 h with agitation. The presence of bound human IgG antibodies was detected using reaction with catalyzed Signal Amplification (CSA) System II, Biotin-Free, HRP, DAB+ (Dako, Agilent, Cat#: K1497), diluted in the substrate buffer (1:100). Results were visualised using Ettan DigeImager (GE Healthcare Life Sciences) and signals (image colour-intensities) quantified using ImageQuant software version 8.1 (GE Healthcare Life Sciences).

Protein oxidation analysis was performed by OxiProteomics (www.oxiproteomics.fr). Extracted proteins were quantified by the Bradford method and split into equal amounts for analyses. Carbonylated proteins were labeled with specific functionalized fluorescent probes, and samples were resolved high‐resolution electrophoresis separation. Total proteins were post‐stained with SyproRuby™ protein gel stain. Image acquisition for carbonylated and total proteins was performed using the Ettan^® DIGE imager (GE Healthcare). Image processing and analysis was performed using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997‐B014). Density histograms and lane profile plots were obtained from each sample, both for carbonylated and total proteins. Carbonylated protein signal was normalized by total proteins signal for each sample in order to obtain their Carbonyl score. Statistical analyses were accomplished using GraphPad Software (La Jolla, California, USA).