Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting.

It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.

Sexual dimorphism in clock genes expression in human adipose tissue.

BACKGROUND: This study was carried out to investigate whether sex-related differences exist in the adipocyte expression of clock genes from subcutaneous abdominal and visceral fat depots in severely obese patients. METHODS: We investigated 16 morbidly obese patients, eight men and eight women (mean age 45 ± 20 years; mean BMI 46 ± 6 kg/m(2)), undergoing laparoscopic gastric bypass surgery. Biopsies were taken as paired samples [subcutaneous and visceral adipose tissue (AT)] at the beginning of the surgical process at 11:00 h in the morning. Metabolic syndrome features such as waist circumference, plasma glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also studied. The expression of clock genes (PER2, BMAL1, and CRY1) was measured by quantitative real-time PCR, Western blot, and immunohistochemical analysis. RESULTS: Gene expression was significantly higher in women than in men for the three genes studied in both ATs (P < 0.05). In visceral fat, these differences were more marked. (P < 0.001). Western blot analysis partially confirmed these results since statistical differences were observed for PER2 in both ATs and for CRY1 in subcutaneous adipose tissue. There were no differences in BMAL1 protein expression. Interestingly, clock gene expression level was correlated with LDL-C and HDL-C (P < 0.05). Moreover, we found significant associations with body fat mass in women and with age in men. CONCLUSIONS: Clock genes expression is sex dependent in human adipose tissue from morbidly obese subjects and correlates to a decreased in metabolic syndrome-related traits. These preliminary results make necessary to go deep into the knowledge of the molecular basis of the sexual dimorphism in chronobiology.