Blots were analyzed by densitometry using Fusion-Capt Software. Sham: sham operated control (n = 8) IA: ischemic/ infarct area RA: remote area. (E, F) Western blot and quantitative analysis of β-tubulin, β-actin and GAPDH protein levels in heart tissue from mouse model of different time point (1 d after surgery, n = 6 4 d after surgery, n = 6 7 d after surgery, n = 6). (C, D) Western blot and quantitative analysis of β-tubulin, β-actin and GAPDH protein levels in heart tissue from Rhesus monkey model of myocardial infarction (4 months after surgery, n = 4). (A, B) Western blot and quantitative analysis of β-tubulin, β-actin and GAPDH protein levels in heart tissue from Rhesus monkey model of early stages of myocardial ischemia (2 h after surgery, n = 2 24 h after surgery, n = 2). Protein levels of β-tubulin, β-actin and GAPDH in the heart tissue from animal model of myocardial ischemia.
GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.ĬBB, Coomassie brilliant blue EDTA, ethylene diamine tetraacetic acid GAPDH, glyceraldehyde-3-phosphate dehydrogenase H&E, hematoxylin and eosin HKP, housekeeping protein Housekeeping proteins LAD, left anterior descending coronary artery Loading control Myocardial ischemia PAGE, polyacrylamide gel electrophoresis PVDF, polyvinylidene fluoride SDS, sodium dodecyl sulfate TBS, Tris-buffered saline TBST, TBS with Tween Total protein Western blot. 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.
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