Computational protocol: Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

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Protocol publication

[…] Due to a limited amount of biological material, Western blot analyses were performed only on duodenal iron transporters and coupled oxido-reductases (DMT1, FPN1, Dcytb, Hp). After protein extraction from duodenal biopsy samples using RIPA buffer (Sigma-Aldrich) and determination of concentration (Thermo Scientific Pierce BCA Protein Assay Kit; Thermo Fisher Scientific Inc., Rockford, IL, USA), six samples were found to be insufficient for further analysis by Western blot. Due to the small amount of tissue obtained from three of the biopsies, only Hp and DMT1 could be evaluated in these samples.Western blot analysis of DMT1 (IRE variants), Dcytb, FPN1, Hp and actin (loading control) levels was carried out with some modifications as described, in detail, previously []. Proteins separated by SDS-PAGE were blotted onto a 0.2 μm nitrocellulose membrane for 2 hrs at 0.25 A using a MiniProtean II blotting apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% BSA in TBS (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 25 min. (DMT1), 40 min. (ferroportin), 60 min. (Dcytb), 45 min. (Hp) or in 5% non-fat milk in TBS for 20 min. (actin). A solution of 0.1% Tween-20/TBS was used for washing. Washed membranes were incubated with the relevant primary antibody. Goat polyclonal antibodies NRAMP 2 (N-20) and Hephaestin (N-20) against human DMT1 and Hp from Santa Cruz Biotechnology (Santa Cruz, CA, USA), goat polyclonal anti-Cytochrome b reductase 1 antibody (EB06633) against human Dcytb (Everest Biotech, Upper Heyford, UK), rabbit polyclonal antibody MTP11-A against human ferroportin from Alpha Diagnostic International (San Antonio, TX, USA) and mouse monoclonal antibody (A3853) against human actin (Sigma-Aldrich) were used. After incubation (overnight, 4°C), the washed membrane was incubated for 1 hr with corresponding horseradish peroxidase-conjugated secondary antibody. Antibodies were diluted in 5% BSA (non-fat milk)/0.1% Tween-20/TBS. The horseradish peroxidase-conjugated secondary antibody was detected by enhanced chemiluminescence using Supersignal reagent from Pierce (Rockford, IL, USA) and a LAS 1000 CCD (Fuji). Band intensities were quantified by densitometry and the data were analysed with the use of ImageJ software (version 1.42q; NIH, USA, available on [...] Data are expressed as mean ± S.E.M. Comparison of the data between groups was performed using the ANOVA and LSD test. When the variables were not normally distributed, the Mann-Whitney test or the Kruskal-Wallis test followed by multiple comparison tests, were used as appropriate. Correlation was assessed using the Spearman rank method. Statistical analysis was done using the Statistica program (version 9; StatSoft, Tulsa, OK, USA) and the GraphPad Prism program (version 5.00; GraphPad Software, Inc., San Diego, CA, USA). The significance level was set at 0.05. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Hemochromatosis, Liver Diseases, Anemia, Iron-Deficiency
Chemicals Iron