Western blot normalization: Time to choose a proper loading control seriously.

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, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (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 sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation 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.

Lactobacillus johnsonii enhances the gut barrier integrity via the interaction between GAPDH and the mouse tight junction protein JAM-2.

Commensal intestinal microbiota interacts with gut epithelial cells in the host by binding to specific host receptors. Several pattern recognition receptors on the gut that sense conserved microbial-associated molecular patterns have been reported; however, many of the gut receptor molecules involved in bacterial binding have not yet been identified. In this study, commensal intestinal bacteria interacting with mouse gut surface proteins were screened from fecal bacterial samples, to identify novel receptors on the epithelial cells in the mouse gut. Among the screened intestinal lactic acid bacteria, the frequently isolated Lactobacillus johnsonii MG was used for the purification of gut receptor proteins. An approximately 30 kDa protein was purified using affinity resin coupled surface layer proteins isolated from L. johnsonii MG. The purified gut protein was identified as a member of the tight junction protein family, junctional adhesion molecule-2 (JAM-2). As expected, the tight junctions of Caco-2 cells damaged by H2O2 were repaired by incubation with L. johnsonii MG. RNA sequence analysis showed significant upregulation of the expression of genes for tight junctions, anti-inflammatory effects, transcriptional regulation, and apoptosis in Caco-2 cells, following L. johnsonii MG treatment. In L. johnsonii MG, the surface layer 40 kDa protein was purified with gut protein-coupled affinity resin and identified as the moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These results suggest that L. johnsonii MG promotes the barrier function integrity in Caco-2 cells via GAPDH-JAM-2 binding. Here, we propose a promising approach to identify novel gut receptor molecules based on commensal bacterial interactions and understand host-bacterial communication in a mouse model.

Total protein staining with Congo red as an alternative loading control for western blot analysis.

For western blot analysis, a housekeeping protein, such as ß-actin or glyceraldehyde-3-phosphate dehydrogenase, is used as loading control with the assumption that these proteins are stable. In practice, these internal loading control proteins vary with different cell states and tissue types. These internal standards are not appropriate for use with serum, extracellular secretion, cerebrospinal fluid analysis or for protein purification. We investigated total protein measurement using Congo red staining and found it to be a superior alternative to routine loading controls. Advantages include lower cost, technical simplicity and improved linear regression. We propose using Congo red staining for total protein immunoblotting to evaluate protein loading in western blots.

First report of Trypanosoma dionisii (Trypanosomatidae) identified in Australia.

Trypanosomes are blood-borne parasites that can infect a variety of different vertebrates, including animals and humans. This study aims to broaden scientific knowledge about the presence and biodiversity of trypanosomes in Australian bats. Molecular and morphological analysis was performed on 86 blood samples collected from seven different species of microbats in Western Australia. Phylogenetic analysis on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences identified Trypanosoma dionisii in five different Australian native species of microbats; Chalinolobus gouldii, Chalinolobus morio, Nyctophilus geoffroyi, Nyctophilus major and Scotorepens balstoni. In addition, two novels, genetically distinct T. dionisii genotypes were detected and named T. dionisii genotype Aus 1 and T. dionisii genotype Aus 2. Genotype Aus 2 was the most prevalent and infected 20.9% (18/86) of bats in the present study, while genotype Aus 1 was less prevalent and was identified in 5.8% (5/86) of Australian bats. Morphological analysis was conducted on trypomastigotes identified in blood films, with morphological parameters consistent with trypanosome species in the subgenus Schizotrypanum. This is the first report of T. dionisii in Australia and in Australian native bats, which further contributes to the global distribution of this cosmopolitan bat trypanosome.

Meat Color Stability of Ovine Muscles Is Related to Glycolytic Dehydrogenase Activities.

The relationship between glycolytic dehydrogenase, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactic dehydrogenase (LDH), and meat color stability was studied in this study using ovine muscle. Three different ovine muscles, including M. longissimus lumborum (LL), M. semimembranosus (SM), and M. psoas major (PM), were obtained (n = 10, respectively), and then displayed for 7 days at 4 °C. The LL and SM muscle had higher surface redness, higher (P < 0.05) GAPDH activity, nicotinamide adenine dinucleotide (NADH) content, and lower (P < 0.05) LDH-B activity than PM muscles during display. The PM muscle had the worst color stability and lowest NADH content. These results suggest that variation in color stability of physiologically different muscles may be affected by glycolysis dehydrogenases. Comparatively, our data showed that GAPDH may play a more important role than LDH-B to maintain meat color stability.

Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots.

Western blotting is an analytical method widely used for detecting and (semi-)quantifying specific proteins in given samples. Western blots are continuously applied and developed by the protein community. This review article focuses on a significant, but not yet well-established, improvement concerning the internal loading control as a prerequisite to accurately quantifying Western blots. Currently, housekeeping proteins (HKPs) like actin, tubulin, or GAPDH are often used to check for equal loading or to compensate potential loading differences. However, this loading control has multiple drawbacks. Staining of the total protein on the blotting membrane has emerged as a better loading control. Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5-10 µg. Although these and further significant advantages have been demonstrated over the past 10 years, only a small percentage of laboratories take advantage of it. The objective of this review article is to collect and compare information about TPS options and to invite users to reconsider their applied loading control. Nine benefits of TPS are discussed and seven different variants are critically evaluated by comparing technical details. Consequently, this review article offers an orientation in selecting the appropriate staining type. I conclude that TPS should be the preferred loading control in future Western blot approaches.

Profiling protein expression in circulating tumour cells using microfluidic western blotting.

Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

Proteomic analysis of coffee grains exposed to different drying process.

Many biochemical events occur inside grains during post-harvest processes. Several methods have been developed to relate the chemical composition of the coffee grain to the beverage quality, including identification of possible molecular markers for flavor characterizing. This study was aimed at evaluating the changes in the proteomic profile of pulped and natural C. arabica grains dried in a yard or dryer at 60°C. It was observed that fruits dried in a dryer at 60°C showed an altered proteomic profile, with a reduction in the most abundant proteins compared to those yard-dried grains. Among the identified proteins, those involved in the metabolism of sugars and stress response were highlighted. Results have shown that post-harvest processes that impact coffee quality are related to changes in protein abundance, indicating that proteomic analysis may be effective in the identification of biochemical changes in coffee grains subjected to different post-harvest processes.

Housekeeping proteins: How useful are they in skeletal muscle diabetes studies and muscle hypertrophy models?

The use of Western blot analysis is of great importance in research, and the measurement of housekeeping proteins is commonly used for loading controls. However, Ponceau S staining has been shown to be an alternative to analysis of housekeeping protein levels as loading controls in some conditions. In the current study, housekeeping protein levels were measured in skeletal muscle hypertrophy and streptozotocin-induced diabetes experimental models. The following housekeeping proteins were investigated: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-actin, α-tubulin, γ-tubulin, and α-actinin. Evidence is presented that Ponceau S is more reliable than housekeeping protein levels for specific protein quantifications in Western blot analysis.

Cell specific differences in the protein abundances of GAPDH and Na(+),K(+)-ATPase in skeletal muscle from aged individuals.

Na(+), K(+)-ATPase (NKA) isoforms (α1,α2,α3,ß1,ß2,ß3) are involved in the maintenance of membrane potential and hence are important regulators of cellular homeostasis. Given the age-related decline in skeletal muscle function, we investigated whether the natural physiological process of aging is associated with altered abundance of NKA isoforms (α1,α2,α3,ß1,ß2,ß3) or of the commonly used control protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, measurements were made in both whole muscle or specific fiber types obtained from skeletal muscle biopsies. Seventeen healthy older (AGED, 69.4 ± 3.5 years, mean ± SD) and 14 younger (YOUNG, 25.5 ± 2.8 years) adults underwent a muscle biopsy for biochemical analyses. Comparing homogenates from AGED and YOUNG individuals revealed higher ß3 isoform (p<0.05) and lower GAPDH (p<0.05). Analysis of individual fibers in muscle from YOUNG individuals, showed greater α3 and ß2 isoforms, and more GAPDH in Type II compared with Type I fibers (p<0.05). In the AGED, GAPDH was higher in Type II compared with Type I fibers (p<0.05), there were no fiber type differences in the NKA isoforms (p>0.05). Compared with the same fiber type in YOUNG, α1 was greater (Type I) and α3 lower (Type II), while in both fiber types, ß2 was lower, ß3 greater and GAPDH lower, in muscle from AGED individuals (all p<0.05). Overall, we demonstrate that (i) GAPDH is an inappropriate choice of protein for normalization in all skeletal muscle research and (ii) full understanding of the role of NKA isoforms in human skeletal muscle requires consideration of age and muscle fiber type.

Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell.

Ion mobility mass spectrometry experiments enable the characterization of mass, assembly, and shape of biological molecules and assemblies. Here, a new radio-frequency confining drift cell is characterized and used to measure the mobilities of peptide, protein, and protein complex ions. The new drift cell replaced the traveling-wave ion mobility cell in a Waters Synapt G2 HDMS. Methods for operating the drift cell and determining collision cross section values using this experimental set up are presented within the context of the original instrument control software. Collision cross sections for 349 cations and anions are reported, 155 of which are for ions that have not been characterized previously using ion mobility. The values for the remaining ions are similar to those determined using a previous radio-frequency confining drift cell and drift tubes without radial confinement. Using this device under 2 Torr of helium gas and an optimized drift voltage, denatured and native-like ions exhibited average apparent resolving powers of 14.2 and 16.5, respectively. For ions with high mobility, which are also low in mass, the apparent resolving power is limited by contributions from ion gating. In contrast, the arrival-time distributions of low-mobility, native-like ions are not well explained using only contributions from ion gating and diffusion. For those species, the widths of arrival-time distributions are most consistent with the presence of multiple structures in the gas phase.

Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include ß-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that ß-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke.

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

[Contribution to the study of glyceraldehyde-3-phosphate dehydrogenase in patients with type 2 diabetes]. / Contribution à l'étude de l'enzyme glycéraldéhyde-3-phosphate déshydrogénase chez des sujets atteints de diabète type 2.

Diabetes is recognized as a major public health problem responsible for early morbidity and mortality with a worldwide prevalence in permanent increase. The type II diabetes once called non-insulin dependent diabetes, accounts for about 90 % of all forms of diabetes and is characterized by abnormalities that affect insulin secretion and insulin action and thus, induces hyperglycemia. The aim of this work is to study the involvement of a key enzyme of glycolysis, glyceraldehyde-3-phosphate dehydrogenase in type 2 diabetes. This work includes a biochemical, kinetic studies, and the study of the expression of GAPDH in subjects with type 2 diabetes. From our study, we could classify the diabetic subjects into two categories: the first one, consisting of subjects in whom GAPDH has a specific activity and an electrophoretic profile similar to healthy subjects, and the second one, in which there is an inhibition of GAPDH. Our results suggest that, in 60 % of our patients with type 2 diabetes, a reversible inhibition of GAPDH is observed. This inhibition is probably mediated by the ionic interaction with the erythrocyte membrane protein, band 3.

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, ß-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.

Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.

Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.

Identification of proteins with increased levels in ameloblastic carcinoma.

PURPOSE: The comparative proteomic approach by a combination of 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MS) analysis is an attractive strategy for the discovery of cancer biomarkers and therapeutic targets. The identification of protein biomarkers associated with ameloblastic carcinoma (AC), a malignant epithelial odontogenic tumor, will potentially improve the diagnostic and prognostic accuracy for this malignant neoplasm. The aim of the present study was to identify highly expressed proteins in AC that could be considered as potential biomarkers. MATERIALS AND METHODS: The protein profile of an AC was compared with the protein profiles of 3 cases of benign ameloblastoma. Proteins that showed increased levels in AC were identified using MS, and the augmented amount of some of these proteins in the malignant lesion was confirmed by Western blot or immunohistochemistry. RESULTS: We detected a total of 782 spots in the protein profile of AC, and 19 of them, showing elevated levels compared with benign ameloblastoma, were identified using MS. These proteins have been implicated in several cellular functions, such as cell structure, metabolism, stress response, and signal transduction. CONCLUSIONS: The increased expression of the identified proteins and the minor expression of some proteins that might inhibit tumor progression could be involved in the evolution from a benign lesion to carcinoma.