Computational protocol: A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice

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Protocol publication

[…] Protein extracts (20 μg) were separated under denaturing conditions on precast 4% to 20% mini gels, transferred to a nitrocellulose membrane, and blocked with 5% non-fat dry milk. Membranes were incubated overnight with rabbit-anti-Glo1 (1 : 1000; ab96032, Abcam, Cambridge, UK) or for one hour with mouse-anti-actin (1 : 5000; MP Biomedicals, Eschwege, Germany). Membranes were subsequently incubated with the appropriate horseradish peroxidase conjugated secondary antibody (1 : 5000; Jackson ImmunoResearch Laboratories, Europe; Dianova, Hamburg, Germany). Immunoreactive proteins were visualized on X-ray films using enhanced chemiluminescence detection reagents, according to the manufacturer's instructions. Densitometric analysis (ImageJ) was used to determine relative Glo1 levels, normalized to actin as a loading control. [...] Statistical analysis was performed using SigmaStat 2.0 software (Jandel GmbH, Erkrath, Germany) or SPSS Statistics version 22 (IBM, Armonk, USA). Experimental values are expressed as mean ± SD unless otherwise stated. Statistically significant differences between groups were calculated by Student's t-test or two-way ANOVA. Values of p < 0.05 were considered significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Nervous System Diseases, Atherosclerosis
Chemicals Pyruvaldehyde