The following day, blots were rinsed in TBST for 6 times, 5 min each, and incubated for 1 h at 37 ☌ with horseradish peroxidase conjugated rabbit anti mice secondary antibodies (at 1:2000 dilution, Sigma A9044, USA). The blots were then incubated with primary antibodies anti-β-actin, GAPDH, and β-tubulin, (at 1:1000 dilution, ZSGB-BIO, China) overnight at 4 ☌.
Membranes were blocked in Tris-buffered saline with Tween solution (TBST, 50 mM Tris-HCl, PH 8.0, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk for 1 h at room temperature. Equal amounts of protein from each sample (30 μg) and pre-stained protein marker (26616, SM0671 26619, SM1811, Thermo, USA) were separated by 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride membrane (PVDF, Bio-Rad Laboratories) under electrophoretical conditions (300 mA, 2 h). Protein samples were mixed with 5× loading buffer, denatured in boiling water for 10 min, then cooled and stored at −20 ☌ for further study. The supernatant was separated by centrifugation at 12,000 g for 15 min at 4 ☌ and total protein concentration was detected by BCA protein assay kit (Thermo Pierce, USA) using bovine serum albumin as standard. Heart tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40%, and 0.1% SDS), supplemented with 1% complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannhein, Germany). And it can be applied practically in other studies related to ischemic heart disease. In our study, we found that the appropriate loading control for western blot analysis of ischemic myocardium in Rhesus monkey and mouse models of ischemic heart disease is total protein level. In the present study, a mouse model was used for its common employment in the study, and a Rhesus monkey model was used for its similarity to human disease,. This makes the selection of loading control for western blot analysis of the ischemic tissue a challenge. The cell type and proteins expressed in the ischemic heart are usually different among various stages after the onset of disease. Ischemic heart disease is typically caused by blockage of coronary artery, which leads to the loss of vital components in the heart, such as cardiomyocytes, resulting in myocardial infarction and eventual cardiac dysfunction or heart failure. Under these circumstances, loading controls should be cautiously selected. However, studies have shown that expression levels of these HKPs were varied in some experimental conditions or diverse samples,. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin have been extensively used as loading controls on the account of their expression levels are generally assumed to be insensitive to the influence of various physiological conditions and treatments. The loading controls should have a constant expression level regardless of experimental conditions. In order to quantify the expression levels of the target proteins in various samples, loading controls are commonly used as internal standards. Western blot has been an essential technique for detecting the relative expression of proteins in different samples (such as cells, tissues, etc.) since the first publication in 1979. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey. We concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. However, the protein level of GAPDH was stable in the Rhesus monkey model. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The heart tissue samples from different areas and time points after surgery were subjected to western blot or gel staining. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. This study was undertaken to look for an appropriate loading control for western blot analysis of ischemic myocardium. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction.
Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. An appropriate loading control is critical for Western blot analysis.
0 kommentar(er)
