Computational protocol: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

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Protocol publication

[…] Whole-cell lysates were prepared using complete Lysis-M buffer (Roche Diagnostics) and protein content determined using a protein assay kit (Pierce). Equal amounts of protein (50 μg) were resolved using 13% SDS -PAGE under reducing condition and electro-transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, CA). Membranes were blocked with 5% skim milk then incubated with Cygb antibody (1:60 rabbit anti-human polyclonal antibody [sc-66855] Santa Cruz Biotechnology, Inc, CA) or α-tubulin antibody (1:5000, mouse anti-human α-tubulin monoclonal antibody, SIGMA) as loading control. Membranes were incubated with polyclonal goat anti-rabbit IgG (H+L) horseradish peroxidase (HRP)-conjugated (1:25000, Jackson ImmunoResearch Laboratories, Inc. PA) or polyclonal goat anti-mouse IgG HRP-conjugated (1:6000, DakoCytomation Denmark A/S, Demark). Bound proteins were detected using chemiluminescence reagents (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, IL) and visualized by exposing to X-ray film (Fuji Photo Film, Japan) that was developed using a Kodak X-OMAT 2000A processor (Eastman Kodak Company, Japan). X-ray films were scanned using an Artixscan 1800f scanner (MICROTEK, TAIWAN) and bands were analyzed using Quantity One 1-D analysis software (Bio-Rad Laboratories). The integrated areas of bands were determined and expressed in arbitrary units (AU). [...] Data from four replicate experiments were expressed as mean ± standard error. Statistical analyses were performed using SigmaPlot 11 software (Systat Software, Inc). Differences between groups were compared using one-way ANOVA or ANOVA on ranks (Kruskal-Wallis) based on the normality and equal variance tests. To determine exactly which groups are different and the size of the difference, multiple comparisons versus control group were carried out using Bonferroni t-test and Dunnett's or Dunn's test for one-way ANOVA and ANOVA on ranks (Kruskal- Wallis), respectively, as post-hoc tests. […]

Pipeline specifications

Software tools Quantity One 1-D, SigmaPlot
Applications Miscellaneous, DNA fingerprinting
Organisms Homo sapiens
Diseases Brain Neoplasms, Glioblastoma, Neoplasms