nitrocellulose membrane sheets  (Bio-Rad)

Journal: Biomolecules

Article Title: Central Actions of Leptin Induce an Atrophic Pattern and Improves Heart Function in Lean Normoleptinemic Rats via PPARβ/δ Activation

doi: 10.3390/biom14081028

Figure Lengend Snippet: Effect of central leptin on PPARβ/δ protein levels in the heart. Western-Blot analysis of PPARβ/δ in 50 µg of total extracts from cardiac ventricles after central saline or leptin infusion. The values are expressed relative to the SS group. Data are the mean ± SEM (n = 5) per group of animals. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Different letters indicate significant differences ( p ≤ 0.05). Groups: SS: saline-infused rats fed ad libitum ; PF: saline-infused pair-fed rats; Lep: leptin-infused rats; Lep+GSK0660: leptin-infused rats and treated with GSK0660. A representative Red Ponceau staining of the nitrocellulose membrane for total protein normalization prior to immunodetection and the quantitative densitometry readings of PPARβ/δ protein from the Western-Blot analysis are depicted in .

Article Snippet: Samples were previously mixed with SDS sample buffer and boiled at 95 °C for 10 min. Proteins were transferred to nitrocellulose sheets (0.2 μm, Bio-Rad, Hercules, CA, USA) and incubated overnight (12–16 h) at 4 °C with the appropriate primary antibodies, followed by incubation at room temperature for 2 h with corresponding secondary antibody conjugated with horseradish peroxidase.

Techniques: Western Blot, Saline, Staining, Membrane, Immunodetection