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Absorbance techniques

  • Total protein concentration quantification in tissue or plasma samples using Protein Assay (BioRad)
  • Protein oxidative damage analysis through total carbonyls determination in plasma samples using dinitrophenylhydrazine(DNPH)-based assay
  • Lipid oxidative damage analysis through malondialdehyde (MDA) determination in plasma samples using thiobarbituric acid (TBARs) assay adapted to microplate reader
  • Plasma thiol groups (-SH) quantification using 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB) assay adapted to microplate reader 
  • Superoxide dismutase (SOD) activity using a colorimetric assay (Invitrogen)
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Analysis with 2300 EnSpire Multimode Plate Reader (PerkinElmer)

Fluorescence techniques

  • Reduced glutathione (GSH) content quantification in plasma samples by a fluorimetric method based on the reaction with o-phthaldhyde (OPA) adapted to microplate reader 
  • Xanthine oxidase activity (XOD) quantification  in plasma samples by a a fluorimetric method based on the reaction with Amplex Red
  • Catalase activity (CAT) quantification  in plasma samples by a a fluorimetric method based on the reaction with Amplex Red
Analysis with 2300 EnSpire Multimode Plate Reader (PerkinElmer)

Luminescence techniques

  • Total Antioxidant Capacity (TAC) for the analysis of low-molecular-weight antioxidants contained in plasma samples using a chemoluminescence inhibition assay based on horseradish peroxidase-catalyzed luminol
  • Superoxide anion scavenger activities (SOSA) in plasma samples using a coelenterazine-based luminescence assay
  • MMP-9-TIMP-1 and MMP-2-TIMP-2 protein interactions in plasma samples using amplified luminescent proximity homogeneous assay (ALPHA)-LISA (PerkinElmer)
Analysis with 2300 EnSpire Multimode Plate Reader (PerkinElmer)

ELISAs

  • MMP-9, MMP-2, TIMP-1 and TIMP-2 concentration quantification using commercial quantikine ELISA kits (R&D Systems)  
  • MMP-9 and MMP-2 activity quantification using commercial ELISA kits (QuickZyme Biosciences)
  • Oxidized LDL determination using commercial ELISA kit (Mercodia)
  • 8-hydroxy-2-deoxy guanosine determination using commercial DNA damage (8-OHdG) ELISA kit (StressMarq Biosciences, INC)
  • Biomarkers of mineral metabolism (calcium, phosphate, vitamin D, PTH, FGF-23, sKL, among others) using different commercial ELISA kits  
Analysis with 2300 EnSpire Multimode Plate Reader (PerkinElmer)

Zymography

  • MMP-9 and MMP-2 gelatinase activity using SDS-PAGE in non-reducing conditions. Bands correspond to gelatinase activity, which is quantified by densitometry using ImageJ  
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Analysis with Mini-PROTEAN 3 (BioRad)

Western blot 

  • Expression of different native proteins and in their phosphorylated and oxidized forms using SDS-PAGE in reducing conditions. Bands corresponding to the specific protein expression are quantified by densitometry using ImageJ  
  • Immunoprecipitation and co-immunoprecipitation of proteins using protein A and G sepharose beads (GE Healthcare) 
​Analysis with Mini-PROTEAN 3 (BioRad)

Immunofluorescence

  • Localization and co-localization of proteins in isolated fixed and permeabilized  cells using antibodies Alexa Fluor 568, 488-conjugated secondary antibodies. Nuclei are stained with DAPI or Hoechst
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​Analysis with Confocal Microscopy with LSM 510 Meta ZEISS with ConfoCor 3 Module

Immunohistochemistry

Detection and localization of proteins in human heart sections by incubation of primary antibody to the target, and subsequent binding of a labelled secondary antibody to the primary antibody.  The secundar antibody can thereby be visualized by a marker such as fluorescent dye.
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