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Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology
Abstract
Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.
... Stain-free image was selected as normalization channel and the normalized volumes are indicated in the analysis Table on the tool bar. The target protein band intensity value is adjusted for variation in the protein total load on the gel, following other studies (28,29). ...
Anti‑androgen drugs are the standard pharmacological therapies for treatment of non‑metastatic prostate cancer (PCa). However, the response of PCa cells may depend on the anti‑androgen used and often patients become resistant to treatment. Thus, studying how the anti‑androgen drugs affect oncogenes expression and action and the identification of the best strategy for combined therapies are essential to improve the efficacy of treatments. The Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an oncogene associated with PCa progression and aggressiveness, although its relationship with the androgen receptor signaling remains to be elucidated. The present study aimed to evaluate the effect of anti‑androgens in regulating STEAP1 expression and investigate whether silencing STEAP1 can make PCa cells more sensitive to anti‑androgen drugs. For this purpose, wild‑type and STEAP1 knockdown LNCaP cells were exposed to bicalutamide, enzalutamide and apalutamide. Bicalutamide decreased the expression of STEAP1, but enzalutamide and apalutamide increased its expression. However, decreased cell proliferation and increased apoptosis was observed in response to all drugs. Overall, the cellular and molecular effects were similar between LNCaP wild‑type and LNCaP‑STEAP1 knockdown cells, except for c‑myc expression levels, where a cumulative effect between anti‑androgen treatment and STEAP1 knockdown was observed. The effect of STEAP1 knockdown alone or combined with anti‑androgens in c‑myc levels is required to be addressed in future studies.
... Densitometric quantification of Western blots was performed by utilizing ImageJ software (NIH). GRHPR protein expression was normalized to loading controls GAPDH and expressed relative to fold change (Neris et al., 2021). ...
Pediatric nephrolithiasis (NL) or Kidney stone disease (KSD) is an untethered topic in Asian population. In Western countries, the annual incidence of paediatric NL is around 6–10%. Here, we present data from West Bengal, India, on lower age (LA, 0–20 years) NL and its prevalence for the first time. To discover the mutations associated with KSD, twenty-four (18 + 6) rare LA-NL patients were selected for Whole Exome Sequencing (WES) and Sanger sequencing, respectively. It was found that GRHPR c. 494G>A mutation (MZ826703) is predominant in our study cohort. This specific homozygous mutation is functionally studied for the first time directly from human peripheral mononuclear cell (PBMC) samples. Using expression study with biochemical activity and computational analysis we assumed that the mutation is pathogenic with loss of function. Moreover, three genes, AGXT, HOGA1 and GRHPR with Novel variants known to cause hyperoxaluria were found frequently in the study cohort. Our study analyses the genes and variations that cause LA-NL, as well as the molecular function of the GRHPR mutation, which may serve as a clinical marker in the population of West Bengal, Eastern India.
... The Western blotting method was performed following previous studies (23,24), and the expression of tight junction proteins Occludin, Claudin, ZO-1, GLP-2, IGF-1 was investigated. Total protein was extracted by using the CWBIO kit, Beijing, China. ...
This study aimed to investigate the effects of dietary squalene (SQ) supplementation on the growth performance of early-weaned piglets. Twenty early-weaned piglets were randomly divided into two groups, the squalene group (SQ) and the control group (CON). The CON group was fed a basal diet, and the SQ group was fed a basal diet with 250 mg/kg squalene. The feeding period lasted 21 days. The results showed that SQ significantly increased the final body weight (FWB, P < 0.05), average daily gain (ADG, P < 0.05), and average daily feed intake (ADFI, P < 0.05) and significantly decreased the F/G ratio (feed intake/gain, P < 0.05) and diarrhea index (DI, P < 0.05). In terms of blood biochemical indicators, SQ significantly increased anti-inflammatory factors such as transforming growth factor-β (TGF-β, P < 0.001), interleukin-10 (IL-10, P < 0.001), and interferon-γ (IFN-γ, P < 0.01), and decreased pro-inflammatory factors such as tumor necrosis factor-α (TFN-α, P < 0.001) and interleukin-6 (IL-6, P < 0.001). Furthermore, SQ significantly increased blood antioxidant indexes (P < 0.001) such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC) and significantly decreased the level of malondialdehyde (MDA) (P < 0.001). The villus height (P < 0.001) and V/C ratio (villus height/crypt depth, P < 0.001) of the jejunum were significantly increased in the SQ group, while the crypt depth (P < 0.01) was decreased compared to the CON group. The intestinal permeability indexes, namely diamine oxidase (DAO), D-lactic acid (D-Lac), regenerative insulin-derived protein 3 (REG-3), and FITC-Dextran 4 (FD4), significantly decreased the concentrations in the treatment group (P < 0.001), and the antioxidant indexes of the jejunum, such as SOD, GSH-Px, CAT, and MDA, were improved by adding SQ. The qPCR results showed that adding SQ could significantly increase the mRNA expression of jejunal tight-junction proteins, such as zonula occludens-1 (ZO-1, P < 0.001), Occludin (P < 0.001), Claudin (P < 0.001), glucagon-like peptide-2 (GLP-2, P < 0.001), and insulin-like growth factor-1 (IGF-1, P < 0.001). Then, we used Western blotting experiments to further confirm the qPCR results. In addition, it was found that adding SQ increased the abundance of beneficial bacteria such as Gemmiger (P < 0.01) and decreased the abundance of harmful bacteria such as Alloprevotella (P < 0.05), Desulfovibrio (P < 0.05), and Barnesiella (P < 0.05). It was interesting that there was a very close correlation among the fecal microbes, growth performance parameters, intestinal barrier, and blood biochemical indicators. In conclusion, the data suggest that SQ supplementation could effectively improve the growth performance of early-weaned piglets by improving the gut microbiota, intestinal barrier, and antioxidant capacity of the blood and jejunal mucosa.
... For each Western blot, ultraviolet activation of the Criterion stain-free gel was used to assess total protein loading for each sample. Band densitometry was performed Accepted Article using Image Laboratory (Version 5.0, Bio-Rad), and the intensity of each protein band was normalized to the total protein [45] and β-actin (a housekeeping protein) in the individual sample. For quantification and to ensure standardization across blots, the expression of the target protein was normalized to the mean value for the control group on the blot, and then all the normalized values were statistically compared to assess the effect of the treatment groups as described previously [23,31,44]. ...
The mechanisms through which the androgen-dependent activation of the androgen receptor (AR) regulates gravid uterine ferroptosis remain unknown. We show that while co-exposure of pregnant rats to the androgen 5α-dihydrotestosterone (DHT) and insulin (INS) triggered uterine ferroptotic signaling cascades, additional treatment with the anti-androgen flutamide increased expression of the key ferroptosis-inhibitory proteins SLC7A11, GSH, and GPX4, reduced iron content, normalized levels of ferroptosis-associated Tfrc, Fpn1, and Ho1 mRNAs, reduced levels of proteins modified by 4-HNE (a marker of ferroptosis), and restored protein levels of NRF2, a key transcription factor regulating antioxidant defense signaling, in the gravid uterus. Furthermore, exposure to DHT alone increased uterine ferroptosis, and NRF2 abundance was negatively correlated with AR status. Co-immunoprecipitation and Western blot assays revealed that the AR physically interacted with endogenous NRF2, and this interaction was increased by DHT exposure in vivo. Our results suggest that AR overactivation and NRF2 suppression cooperate in the regulation of NRF2-targets in uterine ferroptosis.
Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, while no statistically significant changes were observed in the expression of genes relative to GAPDH. Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that SDS and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the CV for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research. This article is protected by copyright. All rights reserved.
The prevalence of non-alcoholic fatty liver diseases (NAFLD) has reached epidemic levels during recent years and a major driver of NAFLD are diets high in fat and fructose. A common practice in the treatment of NAFLD are life-style interventions including for example increased physical activity. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) has been shown to be central in mediating the beneficial effects of exercise training by regulating the expression of key metabolic genes. However, the significance of hepatic PGC-1α for the high fat high fructose (HFFD) induced condition of NAFLD has not been elucidated. Therefore the aim of the present study was to investigate the effect of hepatic PGC-1α on HFFD and exercise-induced changes in the hepatic transcriptome and metabolome in mice. Using gene-arrays and ¹H NMR spectroscopy, the liver transcriptome and metabolome of liver-specific PGC-1α knock-out mice receiving either standard chow, HFFD or HFFD + exercise (HFFD+Ex) were determined. In total 122 genes were identified as differently expressed in mice receiving HFFD for 13 weeks compared to chow, while the loss of hepatic PGC-1α only had very minor effects on the transcriptome. The same was observed for the liver metabolome. The effect of 4 weeks exercise training in combination with 13 weeks of HFFD, had small effects on the transcriptome compared to HFFD alone, while more effects were observed for the metabolome. Together our results highlights a minor regulatory effect of hepatic PGC-1α on the liver transcriptome during high fat high fructose diet and exercise training.
Antimicrobial peptides (AMPs) have proven to inhibit a variety of pathogens. Chromogranin A-N12 (CGA-N12) is a kind of AMP, and it is characterized by stable structure, high anti-Candida activity, and good safety. However, it remains unclear whether CGA-N12 could effectively inhibit the growth of Candida albicans (C. albicans). Colony forming assays were used to measure minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), and time-kill curve. Disseminated C. albicans rabbit model was established to investigate the influence of CGA-N12 on histological damage. The protein and mRNA levels of suppressor of cytokine signaling 1 (SOCS1) after treatment were investigated. The MIC and MFC of CGA-N12 against C. albicans was 6 mg/mL. CGA-N12 considerably inhibited germ tube formation of C. albicans. The fungal load in the tissues and inflammatory factors in the serum were suppressed by CGA-N12. CGA-N12 significantly reduced the histological changes caused by C. albicans, and the protein and mRNA levels of SOCS1 were markedly inhibited. The inhibition effect of CGA-N12 on C. albicans and significant improvement of histological damage by CGA-N12 through microRNA-155/SOCS1 axis were proved in this study. This study proposes a novel therapeutic strategy for the treatment and prevention of C. albicans.
Abbreviations: AMPs: Antimicrobial peptides; MIC: Minimal inhibitory concentration; MFC: Minimal fungicidal concentration; AIDS: Acquired immune deficiency syndrome; PBS: Phosphate buffer saline; FBS: Fetal bovine serum; ROS: Reactive oxygen species; CFU: Colony formation unit; CGA: Chromogranin A; SOCS1: Suppressor of cytokine signaling 1; SDA: Sabouraud Dextrose Agar; GRAVY: Grand average of hydropathicity; C. parapsilosis: Candida parapsilosis; C. albicans: Candida albicans
The serum and glucocorticoid-induced kinase 1 (SGK1) stimulates aldosterone-dependent renal Na ⁺ reabsorption and modulates blood pressure. In addition, genetic ablation or pharmacological inhibition of SGK1 limits the development of kidney inflammation and fibrosis in response to excess mineralocorticoid signaling. In this work we tested the hypothesis that a systemic increase in SGK1 activity would potentiate mineralocorticoid/salt-induced hypertension and kidney injury. To that end, we used a transgenic mouse model with increased SGK1 activity. Mineralocorticoid/salt-induced hypertension and kidney damage was induced by unilateral nephrectomy and treatment with deoxycorticosterone acetate and NaCl in drinking water for six weeks. Our results show that while SGK1 activation did not induce significantly higher blood pressure, it produced a mild increase in glomerular filtration rate, increased albuminuria, exacerbated glomerular hypertrophy and fibrosis. Transcriptomic analysis showed that extracellular matrix and immune response related terms were enriched in the downregulated and upregulated genes, respectively, in transgenic mice. In conclusion, we propose that systemically increased SGK1 activity is a risk factor for the development of mineralocorticoid-dependent kidney injury in the context of low renal mass and independently of blood pressure.
Housekeeping proteins are essential endogenous controls for normalization as they are expected to be stably expressed. However, the stability of the expression level of housekeeping proteins needs to be assessed considering various experimental conditions. Our study evaluated the degree of variability of 7 commonly used housekeeping proteins with regard to their potential utility as normalizers in 56 pairs of matched colorectal adenocarcinoma (CRC) tissue samples and 6 pairs of hepatocellular carcinoma (HCC) tissue samples using multiple reaction monitoring (MRM) and Western blot analyses. A comprehensive experimental design and strict statistical analysis revealed that the expression levels of these 7 housekeeping proteins were not as stable as expected and they all exhibited upregulations to varying degrees in both the CRC and the HCC tissue samples. Consequently, we verified that using the amount of total protein instead of that of an individual protein can serve as a preferable control for studies of protein expression that require normalization.
The applications of Western/immuno-blotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e. resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate and troubleshoot the WB process, to produce reproducible and reliable blots.
It is currently routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-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.
Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts; however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript, we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.
Semi-Quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain free staining as an alternative to β-actin or the protein stain ponceau S showed that Stain free staining was superior to β-actin and as good as or better than ponceau S staining as a loading control for Western blots.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, protein, and enzyme activity levels in hindlimb muscles of adult and senescent Fischer 344 x Brown Norway rats were investigated. Soleus muscles from adult and senescent rats had similar levels of GAPDH. In contrast, muscles containing a large proportion of glycolytic fibers had lower GAPDH levels in senescent rats relative to these muscles in adult rats; this was observed at both the mRNA and protein levels. These data indicate that skeletal muscle glycolytic capacity of fast muscles is diminished with age and that it may be caused by changes at the level of transcription. Also, because GAPDH mRNA levels change with age in several rat muscles, GAPDH mRNA is not always a proper internal control for mRNA analyses of aging skeletal muscle.
Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 and 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used.
Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing.
Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western Blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, the use of three widely used reference proteins (β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin), as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intra-individual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy and different muscle fiber type composition respectively. A stronger linearity of SF and β-actin compared to GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared to young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared to the existing methods for correcting for loading inaccuracy in Western Blotting of human skeletal muscle in applied physiology.
Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.
Beta-actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.
In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons:
Low price
Visible with the eye
Desk top scanners can be employed for image acquisition
Better for quantitative analysis than silver staining
Possible modifications for fast or highly sensitive staining
Mass spectrometry compatible
Western blot is a widely used method for determining specific protein levels. To control and correct for loading error, an internal control is often used. To date, two housekeeping geneâcoded proteins (i.e., beta-actin and beta-tubulin) are widely used as internal controls in the Western blot analysis. However, no information is available concerning the stability of their expressions in response to a traumatic injury to the central nervous system (CNS). If so, their use as an internal control may have a negative impact on data acquisition, analysis, and interpretation. Using Western blot analysis, we demonstrated that spinal cord injury (SCI) induced a significant increase in beta-actin expression which peaked at 7 days post-SCI (2.48-fold). Coefficient of variation (CV) analysis showed that the CV of beta-actin expression was 43.79 +/- 4.67%, significantly higher than that of six loadings from a single sample (6.5 +/- 0.9%, p < 0.01), indicating that increased expression of beta-actin was a result of SCI, instead of a loading error. In contrast, no statistically significant difference was found in beta- tubulin expression following SCI, compared with sham-operated controls. The CV of beta-tubulin expression following SCI was 14.3 beta 3.96%, significantly less than that of the beta-actin expression (43.79 +/- 4.67%; p < 0.01). Taken together, our study suggests that beta-actin whose expression increases following SCI is not a suitable internal control for Western blot analysis of spinal cord tissues following a traumatic injury. In contrast, beta-tubulin, whose expression was not significantly affected by SCI, is a better choice for the internal control.
Protein stains to detect antigen on membranes
- A Dsouza
- RH Scofield