Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting

It is currently a 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.     

Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.     

SELECTION OF INTERNAL CONTROL GENE FOR EXPRESSION STUDIES IN PORPHYRA HAITANENSIS (RHODOPHYTA) AT DIFFERENT LIFE‐HISTORY STAGES1

Accurate gene quantification depends on the use of an appropriate internal control gene, which should be verified before its use for normalizing data. Housekeeping genes, which are expressed at relatively constant levels, are generally regarded as candidate internal control genes. To determine the ideal internal control for gene expression profiles for Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) at different life‐history stages, we used absolute quantification to assess the expression levels of six housekeeping genes (18S ribosomal RNA, 30S ribosomal protein, glyceraldehyde‐3‐phosphate dehydrogenase, elongation factor 3, alpha‐tubulin, and beta‐tubulin) at the sporophyte and gametophyte stages. Housekeeping genes were selected by comparing the differences of observed copy numbers in sporophytes and in gametophytes. TubB (beta‐tubulin) was found to be the optimal internal control gene, because it showed the smallest difference of gene expression. Compared with TubB, other housekeeping genes had greater variation of expression to different degrees.     

Coomassie staining as loading control in Western blot analysis

In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting.     

Proteomic profiling of PrP27‐30‐enriched preparations extracted from the brain of hamsters with experimental scrapie

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP^TSE) of the host‐encoded cellular prion protein (PrP^C). PrP^TSE has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi‐step proteomic approach for the identification of proteins that co‐purify with the protease‐resistant core of PrP^TSE (PrP27‐30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin‐dependent protein kinase α type II, apolipoprotein E, and tubulin as the major components associated with PrP27‐30 but also trace amounts of actin, cofilin, Hsp90α, the γ subunit of the T‐complex protein 1, glyceraldehyde 3‐phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin‐dependent protein kinase α type II, Hsp90α) may associate with PrP^TSE fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP^TSE, whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.     

Cy5 total protein normalization in Western blot analysis

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence.     

Proteomic Analysis of the Response of Liangyoupeijiu （Super High-Yield Hybrid Rice） Seedlings to Cold Stress

Liangyoupeijiu is a super high-yield hybrid rice. Despite its advantages with respect to yield and grain quality, it is sensitive to cold, which keeps it from being widely cultivated. We subjected Liangyoupeijiu seedlings to 4 ℃ cold treatment, then extracted the leaf proteins. After 2-D gel electrophoresis separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, a series of differentially displayed proteins were identified. Some metabolism-associated proteins were found among the downregulated proteins, such as carbamoyl phosphate synthetase, transketolase 1, dihydrolipoamide dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase. The upregulated proteins included both stress-resistance proteins such as nucleoside diphosphate kinase I and proteins that are negative for rice growth, such as FtsH-like protein, plastid fusion and/or translocation factor （Pftf） and actin. Our results indicate that cold may inhibit Liangyoupeijiu growth through decreasing metabolic activity and damaging cell structure.     

Proteomic changes related to "bewildered" circulating platelets in the acute coronary syndrome

Acute coronary syndromes (ACS) are associated with platelet activation. The aim of the present study was to study the protein expression level associated with glycolysis, oxidative stress, cytoskeleton and cell survival in platelets obtained during an ACS. Platelets from 42 coronary ischemic patients, divided into patients admitted within 24 h after the onset of chest pain (ACS group; n=16) and patients with stable coronary ischemic disease (CAD, n=26), were analyzed using proteomics. The expression levels of proteins involved in cellular cytoskeleton (F‐actin capping, β‐tubulin, α‐tubulin isotypes 1 and 2, vinculin, vimentin and two Ras‐related protein Rab‐7b isotypes), glycolysis pathway (glyceraldehyde‐3‐phosphate dehydrogenase, lactate dehydrogenase and two pyruvate kinase isotypes) and cellular‐related antioxidant system (manganese superoxide dismutase) and even the expression and activity of glutathione‐S‐transferase were significantly reduced in platelets from ACS patients compared to CAD patients. Moreover, reduction in the expression of proteins associated with cell survival such as proteasome subunit β type 1 was also observed in ACS platelets compared with CAD platelets. Principal component and logistic regression analysis suggested the existence of factors (proteins) expressed in the platelets inversely associated with acute coronary ischemia. In summary, these results suggest the existence of circulating antioxidant, cytoskeleton and glycolytic‐“bewildered” platelets during the acute phase of a coronary event.     

Proteomic changes in the crucian carp brain during exposure to anoxia

During exposure to anoxia, the crucian carp brain is able to maintain normal overall protein synthesis rates. However, it is not known if there are alterations in the synthesis or expression of specific proteins. This investigation addresses this issue by comparing the normoxic and anoxic brain proteome. Nine proteins were found to be reduced by anoxia. Reductions in the glycolytic pathway proteins creatine kinase, fructose biphosphate aldolase, glyceraldehyde‐3‐phosphate dehydrogenase, triosephosphate isomerase and lactate dehydrogenase reflect the reduced production and requirement for adenosine tri‐phosphate during anoxia. In terms of neural protection, voltage‐dependent anion channel, a protein associated with neuronal apoptosis, was reduced, along with gefiltin, a protein associated with the subsequent need for neuronal repair. Additionally the expression of proteins associated with neural degeneration and impaired cognitive function also declined; dihydropyrimidinase‐like protein‐3 and vesicle amine transport protein‐1. One protein was found to be increased by anoxia; pre‐proependymin, the precursor to ependymin. Ependymin fulfils multiple roles in neural plasticity, memory formation and learning, neuron growth and regeneration, and is able to reverse the possibility of apoptosis, thus further protecting the anoxic brain.     

Effects of platelets on the protein expression in aortic segments: A proteomic approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor‐α (TNF‐α)‐preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton‐related proteins expression was modified by PRP. However, when PRP was coincubated with TNF‐α pre‐stimulated aortic segments lesser number of cytoskeleton‐related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic‐related proteins. However, in TNF‐α‐preincubated segments the presence of PRP upexpressed glyceraldehyde‐3‐phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF‐α‐preincubated segments the expression of fructose 1,6‐bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α‐chain. In addition, the activity of fructose 1,6‐bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall. J. Cell. Biochem. 111: 889–898, 2010. © 2010 Wiley‐Liss, Inc.     

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA₁₀₀). After the comparison of the protein profiles from MA₁₀₀ fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.     

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

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. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction. This study was undertaken to look for an appropriate loading control 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 different areas and time points after surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. However, the protein level of GAPDH was stable in the Rhesus monkey model. We 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.     

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca²⁺. In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase‐like protein, t‐complex protein, and guanine nucleotide protein G_(o) subunit alpha (G_o alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia‐tolerant species), there are species‐specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.     

Blue Dry Western: Simple, economic, informative, and fast way of immunodetection

The analysis by electrophoresis followed by transfer to membranes and immunodetection (Western blot) is probably the most popular technique in protein study. Accordingly, it is a time- and money-consuming procedure. Here a protocol is described where immunodetection can be accomplished in 30 min. This approach also allows permanent staining of proteins by Coomassie Blue R on the membrane before immune staining with clear background and high sensitivity.     

State‐of‐the‐art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β‐tubulin and β‐actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large‐scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem‐mass‐spectrometry data from 27 human tissues, and suggest 20 ubiquitously‐ and constitutively‐expressed, putative internal‐reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.     

Nucleus‐encoded mRNAs for Chloroplast Proteins GapA,PetA, and PsbO are Trans‐spliced in the Flagellate Euglena gracilis Irrespective of Light and Plastid Function

Euglena gracilis is a fresh‐water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus‐encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans‐spliced and possess spliced leader sequence at the 5′‐end and if trans‐splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde‐3‐phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans‐spliced irrespective of light or plastid function.     

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti‐EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab‐sensitive and ‐resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose‐6‐phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab‐resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia‐inducible factor 1‐associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti‐EGFR therapy in colorectal cancer.     

Enzyme activities of human erythrocyte ghosts: effects of various treatments

Summary Ghosts of human erythrocytes prepared by hypotonic hemolysis were assayed for aldolase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase, and glutathione peroxidase and reductase. Cryptic activity of the enzymes was demonstrated by an increase in activity on dilution with water, which caused fragmentation of the ghosts. Aldolase and glyceraldehyde phosphate dehydrogenase were classed as firmly bound; phosphoglycerate kinase was intermediate; the others were loosely bound. Triton X-100 increased the activities of aldolase, glyceraldehyde phosphate dehydrogenase, and phosphoglycerate kinase. The pH of the medium had little effect upon the firmly bound enzymes but it markedly affected the retention of hemoglobin and the activities of the loosely bound enzymes. The presence of Mg or Ca ions enhanced the retention of hemoglobin and the activity of lactate dehydrogenase and pyruvate kinase, with little effect on aldolase and glyceraldehyde phosphate dehydrogenase. Ghosts diluted in water disintegrated into fragments and tubules or vesicles; Mg or Ca at 1mm afforded protection against this. When ghosts were treated with Triton X-100 and adenosine triphosphate, they contracted to about one-seventh of their volume. The shrunken ghosts had lost a considerable proportion of their cholesterol and protein to the medium.     

A colloidal silver staining--destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.     

Comparison of the characteristics of the immobilized and solubilized glyceraldehyde phosphate dehydrogenase of human erythrocyte membranes.

We studied human erythrocyte membrane associated glyceraldehyde phosphate dehydrogenase in order to determine if a biological form of immobilization of the enzyme altered its kinetic properties. The results indicated that the partially purified solubilized glyceraldehyde phosphate dehydrogenase did not differ, with regard to Km for substrates or pH activity profile, from the same enzyme which was immobilized on the inner face of the erythrocyte plasma membrane.