How To Use Loading Control In Western Blot

Biochem Biophys Rep. 2017 Dec; 12: 108–113.

An advisable loading control for western blot analysis in animal models of myocardial ischemic infarction

Received 2017 May 28; Revised 2017 Aug 25; Accustomed 2017 Sep 7.

Abstract

An appropriate loading control is critical for Western blot analysis. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. Merely HKP expression can exist impacted by sure experimental conditions, such as ischemic myocardial infarction. This study was undertaken to wait for an appropriate loading command for western blot analysis of ischemic myocardium. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. The heart tissue samples from unlike areas and fourth dimension points later surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The full protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. Yet, the protein level of GAPDH was stable in the Rhesus monkey model. Nosotros concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.

Abbreviations: HKP, housekeeping protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LAD, left anterior descending coronary artery; H&East, hematoxylin and eosin; EDTA, ethylene diamine tetraacetic acrid; SDS, sodium dodecyl sulfate; Folio, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBST, TBS with Tween; PVDF, polyvinylidene fluoride; CBB, Coomassie brilliant blue

Keywords: Loading control, Western absorb, Housekeeping proteins, Full poly peptide, Myocardial ischemia

1. Introduction

Western blot has been an essential technique for detecting the relative expression of proteins in different samples (such as cells, tissues, etc.) since the get-go publication in 1979. In order to quantify the expression levels of the target proteins in various samples, loading controls are normally used every bit internal standards [ane]. The loading controls should have a constant expression level regardless of experimental weather condition. Housekeeping proteins (HKPs), such as β-actin [2], glyceraldehyde-three-phosphate dehydrogenase (GAPDH) [3], and β-tubulin [4] have been extensively used every bit loading controls on the business relationship of their expression levels are generally assumed to be insensitive to the influence of diverse physiological conditions and treatments. However, studies have shown that expression levels of these HKPs were varied in some experimental weather or diverse samples [two], [four], [5], [6]. Under these circumstances, loading controls should exist charily selected.

Ischemic heart disease is typically caused by blockage of coronary artery, which leads to the loss of vital components in the middle, such equally cardiomyocytes, resulting in myocardial infarction and eventual cardiac dysfunction or heart failure [vii]. The cell blazon and proteins expressed in the ischemic middle are ordinarily dissimilar amongst diverse stages after the onset of disease. This makes the selection of loading control for western blot analysis of the ischemic tissue a claiming. 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 being disease [8], [9], [10].

In our study, nosotros plant that the appropriate loading control for western blot analysis of ischemic myocardium in Rhesus monkey and mouse models of ischemic heart disease is full poly peptide level. And it tin can exist applied practically in other studies related to ischemic middle disease.

2. Materials and methods

2.ane. Animals

Male Rhesus monkeys, aged 2–3 years and weighed 4.v–half dozen.0 kg, were acclimatized to the laboratory conditions at to the lowest degree one month in the Clan for Assessment and Accreditation of Laboratory Animal Care accredited facility.

Male C57BL/6 mice, aged 7–12 weeks and weighed 20–30 g, were obtained from Beijing Vital River Experimental Animal Convenance and Research Center, a Chinese authorities-accredited experiment animal breeding and research center.

All brute procedures according with the Institutional Animal Care and Use Committee (IACUC) of Sichuan University West China Infirmary follows the guidelines of the U.s.a. National Institutes of Health.

2.2. Induction of myocardial infarction

As described in our previous studies [eleven], [12], all Rhesus monkeys were divided into iii groups: early on phase of myocardial ischemia (2 h after surgery, n = 2, 24 h later on surgery, due north = 2), myocardial infarction (4 months afterwards surgery, northward = 4), and sham-operated controls (due north = 4). Rhesus monkeys in the former two groups were subjected to left anterior descending coronary artery (LAD) ligation to induce myocardial ischemic infarction. The sham-operated controls were subjected to the same surgical procedure except for the LAD apoplexy and ligation. The number of Rhesus monkey sample was very express.

Every bit described in our previous studies [13], [14], [xv], C57BL/6 mice (due north = eighteen) were subjected to LAD ligation to induce myocardial ischemia. The sham-operated controls (n = viii) were subjected to the same surgical procedure except for the LAD occlusion and ligation.

ii.3. Tissue grooming

For early on stage of myocardial ischemia group of Rhesus monkeys, 2 h (n = 2) or 24 h (n = 2) after surgery, the animals were sacrificed to obtain center tissue respectively. For myocardial infarction group and sham-operated controls of Rhesus monkeys, 4 months afterward surgery, the animals were sacrificed to harvest center tissue. As for mice, one d (northward = 6), 4 d (due north = half-dozen), seven d (n = half-dozen) after surgery, they were sacrificed to harvest the heart tissues. Ischemia or infarct area was distinguished from remote area past its pale advent.

All eye tissues were stored in liquid nitrogen for western blot. Another portion was fixed with 4% paraformaldehyde and embedded in paraffin as previously described [16], and so sectioned at 4 mm intervals. The sections were stained with hematoxylin and eosin (H&E) to observe the construction, and with Sirius red to notice collagen deposition [17].

2.4. Western blot assay

Heart tissues were lysed in RIPA buffer (fifty mM Tris-HCl, pH eight.0, 150 mM NaCl, 0.v% sodium deoxycholate, ane% NP-forty%, and 0.1% SDS), supplemented with i% complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannhein, Germany). The supernatant was separated past centrifugation at 12,000 one thousand for 15 min at 4 °C and total poly peptide concentration was detected by BCA protein analysis kit (Thermo Pierce, USA) using bovine serum albumin as standard. Poly peptide samples were mixed with 5× loading buffer, denatured in boiling h2o for 10 min, then cooled and stored at −20 °C for further study. Equal amounts of poly peptide from each sample (30 μg) and pre-stained poly peptide marker (26616, SM0671; 26619, SM1811, Thermo, The states) were separated by 10% SDS-Folio gel, and transferred to a polyvinylidene fluoride membrane (PVDF, Bio-Rad Laboratories) under electrophoretical conditions (300 mA, ii h). Membranes were blocked in Tris-buffered saline with Tween solution (TBST, 50 mM Tris-HCl, PH 8.0, 150 mM NaCl, and 0.ane% Tween-20) containing v% nonfat dry milk for 1 h at room temperature. The blots were so incubated with main antibodies anti-β-actin, GAPDH, and β-tubulin, (at 1:one thousand dilution, ZSGB-BIO, China) overnight at iv °C. The following twenty-four hour period, blots were rinsed in TBST for half-dozen times, 5 min each, and incubated for 1 h at 37 °C with horseradish peroxidase conjugated rabbit anti mice secondary antibodies (at 1:2000 dilution, Sigma A9044, USA). Afterwards beingness rinsed in TBST for 6 times, 5 min each and in Tris-buffered saline (TBS) for 5 min, target proteins were visualized as black bands by using chemiluminescenece horseradish peroxidase substrate (Millipore Corporation, U.s.a.), and analyzed by densitometry using Fusion-Capt Software (VILBER LOURMAT, France).

ii.5. Full protein staining of gel

Correct after electrophoresis, gels were incubated in Coomassie vivid bluish solution (CBB, 0.1% R250, 25% isopropanol, 10% acetic acrid) for 45 min and de-stained in de-stain solution (10% acetic acid, 7% ethanol) overnight, and finally washed with double distilled h2o. Stained gels were imaged on the Fusion scanner and analyzed past Fusion-Capt Software (VILBER LOURMAT, French republic).

two.half dozen. PVDF membrane staining

After immunodetection, PVDF membrane was done past parsing solution (0.19% Glycine, 1% SDS, pH 2.0) for 2 h, and stained past CBB solution for 5 min and de-stained in fifty% methanol solution for twenty min. At last, they were done with double distilled water. Stained PVDF membrane were imaged straight on Fusion scanner and analyzed by Fusion-Capt Software (VILBER LOURMAT, French republic).

ii.7. Statistical analysis

All data are presented equally mean ± SD. SPSS for Windows (USA) was applied to perform the statistical processing. One-style ANOVA was used for statistical evaluation for sham and unlike areas of the myocardial ischemic grouping, P < 0.05 was considered as statistically significant.

3. Results

iii.1. Animal models of ischemic heart illness

H&E staining and Sirius red staining of transversely sectioned heart tissues were used to ascertain the successful establishment of ischemic heart disease animal model (Fig. 1A and B). Infarct area was full of thick and wave shaped collagens, which were stained red by Sirius red.

H&E and Sirius cerise staining of eye tissue. (A) H&E and Sirius reddish staining of transversely sectioned mouse hearts. (B) H&E and Sirius red staining of transversely sectioned Rhesus monkey hearts. The infarct area was full of collagens. Bar = 100 µm. Sham: sham operated command; IA: infarct area; RA: remote area.

3.2. Expression of β-tubulin, β-actin and GAPDH in centre tissues of animal models of myocardial ischemia/ infarction

We detected the protein level of β-tubulin, β-actin and GAPDH in heart tissues by western absorb. Protein level of β-tubulin was various in the eye tissue during different stages of myocardial ischemia/ infarction Rhesus monkey model. In the early stage of ischemia, poly peptide level of β-tubulin decreased in remote area compared with sham control (Fig. twoA and B). On the contrary, β-tubulin was increased in infarct surface area of 4-month myocardial infarction (Fig. 2C and D). In mice model, β-tubulin was increased in ischemic area of every group compared with sham-operated control (Fig. iiEastward and F).

Protein levels of β-tubulin, β-actin and GAPDH in the heart tissue from creature model of myocardial ischemia. (A, B) Western blot and quantitative analysis of β-tubulin, β-actin and GAPDH protein levels in center tissue from Rhesus monkey model of early stages of myocardial ischemia (2 h after surgery, north = 2; 24 h after surgery, n = 2). (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). (Eastward, F) Western blot and quantitative analysis of β-tubulin, β-actin and GAPDH poly peptide levels in heart tissue from mouse model of different time point (one d later surgery, due north = 6; iv d after surgery, due north = 6; 7 d afterward surgery, n = 6). Sham: sham operated control (north = 8); IA: ischemic/ infarct area; RA: remote surface area. Blots were analyzed by densitometry using Fusion-Capt Software. Results are presented every bit means ± SD. Three independent experiments were run for each outcome. *: P < 0.05 versus sham operated control; #: P < 0.05.

Protein level of β-actin was elevated in ischemic monkey heart tissue of early phase (Fig. 2A and B) and iv months subsequently surgery compared with remote area and sham control (Fig. 2C and D). Moreover, information technology was also raised in ischemic myocardium of mice of unlike time points post LAD ligation (Fig. 2E and F).

GAPDH levels were unchanged in the ischemic monkey middle tissue compared with sham control (Fig. 2A - D). Simply in the mouse model, GAPDH was decreased in the ischemic myocardium from all three time points (i d, 4 d, and 7 d) post-surgery (Fig. twoE and F).

3.3. Total protein level in center tissues of animal models of myocardial ischemia/ infarction

In lodge to examination the level of total protein, we stained the gels with CBB directly subsequently electrophoresis. Total protein level was abiding in heart tissues of dissimilar animal models of myocardial ischemia/ infarction (Fig. iii), which indicates that total protein is a good loading control for western blot in our model. Next, nosotros stained PVDF membrane by CBB after immunoblotting instead of gel to reduce the possible influence on the immunodetection of target proteins. Total poly peptide level was also constant on PVDF membrane, and was not influenced by transferring (Fig. 4).

Gel staining image of total protein in the heart tissue from animal model of myocardial ischemia. (A, B, C) Gel staining epitome of total protein in the heart tissue from Rhesus monkey model of early stages of myocardial ischemia (A), Rhesus monkey model of myocardial infarction (B), and mouse model of dissimilar time point (C). Quantitative analysis of total protein was shown in the right panel. Sham: sham operated control (n = 4); IA: ischemic/infarct area; RA: remote area; Grand: pre-stained protein marker. Gels were analyzed past Fusion-Capt Software. Results are presented as means ± SD. Iii contained experiments were run for each result. *: P < 0.05 versus sham operated command; #: P < 0.05.

Staining of total protein level on blots every bit a validated loading control for Western blot in the center tissue from Rhesus monkey model. (A, B) Western blot and quantitative assay of β-actin in the heart tissue from Rhesus monkey model of myocardial ischemia (4 months afterward surgery, due north = 2). (C, D) Total poly peptide level shown by absorb staining, which was done with parsing solution after β-actin detecting. Sham: sham operated control (n = two); IA: ischemic area (n = two); RA: remote area (northward = ii); M: pre-stained protein marker. Blots were analyzed by densitometry using Fusion-Capt Software. Three independent experiments were run for each result.

iv. Discussion

Since the showtime utilize of western blot method as a semi-quantitative analysis of protein in 1979, loading controls were used to normalize the possible errors from the whole process of western blot. An ideal loading control should be abundant in numerous samples and stable to various physiological conditions or treatments. Some HKPs, such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin accept been used as loading controls for a long menstruum.

However, it has been reported that HKP expression levels were varied in some experimental conditions or various samples. Dittmer et al. showed the expression of β-actin was related to the incubation time and it was non stable in MDA-MB-231 cells [ii]. Liu and his colleagues demonstrated that the β-actin level was increased after spinal cord injury [4]. Some other example of varied HKP expression levels is β-actin and GAPDH were shown that their levels were decreased with aging [6]. Martina B reported that the expression of GAPDH was altered subsequently isoprenaline treatment [5]. Therefore, an appropriate loading control is critical for specific written report model. Our present study showed that β-tubulin and β-actin were differentially expressed in heart tissues from dissimilar stages both in Rhesus monkeys model and mice model of myocardial ischemia. The expression level of GAPDH was consistent in Rhesus monkey model, but inconsistent in mice, indicating that GAPDH could exist considered as loading command in western blot assay of Rhesus monkey centre tissues.

Also HKPs, we also tested the full protein level in our models because information technology has been reported to exist a reliable loading control. Total poly peptide staining was first published in 1995 [18], which makes utilise of the signal intensity of the entire loading lane as a loading control [19]. Total poly peptide normalization is more than authentic considering the normalization is washed against the whole lane (multiple proteins), not a unmarried protein. And there is no need to strip and re-immunoprobe blots, reducing the possible error caused by handling the blot. The methods to stain full poly peptide involves staining of gel or blot by Ponceau [20] or CBB [21]. Gel staining has been employed as loading command in some studies [nineteen] however, gel staining could non eliminate errors produced by the transferring procedure. Moreover, gel staining involves incubating and washing gels before detection of target proteins, which increases the gamble of protein degradation and losing. Therefore, absorb staining is a good option of loading control. In our present study, nosotros found that the level of total protein is consequent in different centre tissues past staining gel with CBB. Then we stained blots with CBB after detection of target proteins, and showed that the blot staining is feasible and reliable as loading control in our model, every bit discussed in other experimental conditions [18], [nineteen], [21]. A technology called Stain-Free was developed in 2013 [22], [23], [24] in which a trihalo compound responds to tryptophan residues when exposed to ultraviolet irradiation of certain moving ridge length, thus creating no need of incubating or washing steps of gel. Merely the accuracy of this method is dependent on the amount of tryptophan in samples and requires a special instrument.

In summary, our data illustrated that β-tubulin and β-actin were not stable in fauna models of myocardial ischemic infarction. GAPDH can piece of work as a reliable loading command simply in Rhesus monkeys model of myocardial ischemia. Total protein level is the all-time loading command for western absorb in beast models of myocardial ischemic infarction.

Acknowledgement

This piece of work was supported by National Science Foundation of China (81230004 and 81600214). The authors give thanks Pengfei Han and Qin Sheng for providing the animal models. We also thank Ying Xiao and Ning Wang for pathological technical support.

Appendix A. Transparency document

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