Direct Blue 71 staining as a destaining‐free alternative loading control method for Western blotting

In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining—a novel, sensitive, dye‐binding staining method compatible with immunodetection—may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5–40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein‐based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining‐free alternative loading control method for Western blotting.     

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.     

Β‐actin does not show the characteristics of a reference protein in human cortex

Levels of a reference protein must be the same as a proportion of total protein in all tissues and, in the study of human diseases, cannot vary with factors such as age, gender or disease pathophysiology. It is increasingly apparent that there may be few, if any, proteins that display the characteristics of a reference protein within the human central nervous system (CNS). To begin to challenge this hypothesis, we used Western blotting to compare variance in levels of the “gold standard” reference protein, β‐actin, in Brodmann's area 9 from 194 subjects to variance of total transferred protein measured as intensity of Ponceau S staining. The coefficient of variance of sum intensity measurements for β‐actin levels across all donors was 47% compared to 24 and 27% for the sum intensity of Ponceau S staining measured using two different detection techniques. These data strongly suggest that the level of β‐actin, proportional to total protein, is not constant in human cortex which raises further doubt about the use of reference proteins to normalise data in human CNS studies. Considering our data, we suggest an alternative approach to presenting data from Western blotting of human CNS.     

Fluorescent labeling of proteins with nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots

The fluorescent hydrophobic dye Nile red allows the rapid, sensitive, and general staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Nile red staining does not preclude further electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes. The resulting Western blot can be stained with the covalent fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) using a simple procedure. MDPF staining allows further N-terminal microsequencing and immunodetection of specific bands. This review considers the physicochemical, structural, and analytical studies that have led to the development of Nile red and MDPF staining methods. The usefulness of these procedures is discussed in comparison to other currently available fluorescent and nonfluorescent protein detection methods.     

Simultaneous spectrophotometric determination of Tartrazine, Sunset Yellow and Ponceau 4R in commercial products by partial least squares and principal component regression multivariate calibration methods

Three multivariate calibration methods, partial least squares (PLS-1 and PLS-2) and principal component regression (PCR), were proposed and successfully applied to the simultaneous determination of three dyes, Tartrazine (E-102), Sunset Yellow (E-110) and Ponceau 4R (E-124) in mixtures by ultraviolet-visible absorption spectrophotometry. The designed and optimized training set of calibration was applied to the determination of the three dyes in several synthetic mixtures, containing 1.6–20.0 mg/L of Tartrazine, 3.2–40.0 mg/L of Sunset Yellow and 3.2– 36.0 mg/L of Ponceau 4R. 94.5–105.3% recovery values were obtained. Three commercial foods that contained the three dyes were also satisfactorily analyzed without separation step. The results obtained by the application of the three chemometric approaches in the commercial products are discussed and compared with those obtained by an HPLC method and very similar values were found by all methods. Repeatability and reproducibility studies (with the Student’s and F tests) were achieved over two series of nine standards for each dye, showing no significant differences at the 95% confidence level.     

Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.     

A simple method for assigning multiple immunogens to their protein on a two-dimensional blot and its application to asthma-causing allergens

A "one-step" procedure, that not only removes the color and blocking proteins used in the colorimetric immunodetection step but also stains the proteins originally on the blot, is presented. Following immunostaining and recording of immunoreactive spots, the blot was allowed to air-dry overnight (or longer) at room temperature and then counterstained with a colloidal gold solution. This "air-drying" process apparently altered the affinity of the blocking proteins (and possibly other proteins added subsequently to the blotting step) towards the nitrocellulose membrane causing them to be removed by the acidic colloidal gold solution while the "blotted" proteins were being stained. The sensitivity of this counterstained blot was comparable to that of the blot without going through the immunodetection process. Since both immunodetection and protein staining were carried out on the same blot, this allowed easy identification of many immunoreactive spots to their corresponding proteins when the two profiles were superimposed. Using this procedure, we have detected 25 immunoreactive spots (or allergens) from the whole body extract of the German cockroach (Blattella germanica) that contribute to asthma and assigned them to their corresponding proteins on a two-dimensional (2-D) protein map. The apparent Mr and pI for each of the allergens were determined. We have also located one of the major cockroach allergens, Bla g 5 (glutathione S-transferase). Two-dimensional zymography revealed the presence of ten gelatinase-type proteolytic enzymes. Only one of the ten proteases comigrated with the immunoreactive proteins indicating that unlike other allergen-producing systems, most of the cockroach allergens do not possess protease activity.     

Native red electrophoresis--a new method suitable for separation of native proteins

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state).     

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 β‐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 β‐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.     

Taurine Inhibits Myocardial Fibrosis via PKC-ERK1/2 Signaling Pathways

Previous studies have demonstrated the important role of taurine in inhibiting proliferation of myofibroblasts( myoFb) and myocardial fibrosis. However, the underlying mechanisms are unclear. The present study was designed to shed light on this issue through exploring the signal pathways via in vitro experiments. Angiotension Ⅱ (AngⅡ) treatment significantly increased myoFb proliferation and the levels of collagens Ⅰ and Ⅲ(P<0.05), whereas taurine, PKCα(PKC: protein kinase C) specific inhibitor L-threo-dihydro-sphingosine(D4681), ERK1/2 inhibitor (PD98095) abrogated myoFb proliferation and collagen levels(P<0.05, P<0.01, respectively), and increased the G₀/G₁ phase rate and decreased S phase rate. Immunocytochemistry, confocal fluorescence staining and image analysis showed that taurine could inhibit the translocation and expression of p-PKCαin membrane, and then inhibit nuclear translocation and expression of p-ERK1/2. These results have statistically significant differences compared with those of AngⅡ group(P<0.01). Western blot results also show that taurine could inhibit the protein expression of p-PKCα and p-ERK1/2. We used p-PKCα specific inhibitor D4681 in order to elucidate the relationship between p-PKCα and p-ERK1/2 in signal transduction pathways. Finally, the results show that the protein expression of p-ERK1/2 and nuclear translocation were suppressed in D4681 group.     

Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.     

Validation of Reference Genes for Silver Nanotoxicity Analysis in MCF-7 Cell Line

Following the increasing use of silver nanoparticles (AgNPs) and the growing exposure to these particles, nanotoxicity has become a general concern among researchers around the world. Although quantitative real time PCR (RT-qPCR) is the technique of choice in genotoxicity assessment, use of unconfirmed reference genes could result in data misinterpretation. GeNorm and NormFinder algorithms assist in the selection of appropriate reference genes in RT-qPCR expression analysis. Using these two algorithms, here, the stability of six reference genes’ expression including GAPDH, ACTB, UBC, TBP, HPRT-1 and YWHAZ was evaluated in MCF-7 cells treated with different concentrations (under IC50 estimated by MTT assay) of AgNP. results showed that cytotoxicity can occur in a dose dependent fashion in MCF-7 cells with IC50 value of 5.495 µg/ml. Cumulative analysis by geNorm and NormFinder programs indicated that the normalization of the RT-qPCR data under AgNPs treatment in MCF-7 cells would be greatly improved utilizing two reference genes including TBP and GAPDH. This study is the first investigation for the selection of suitable reference genes for RT-qPCR analysis in MCF-7 cells treated with different concentration of AgNPs, and could be used in future researches established to reveal mechanisms underlying nanosilver exposure.     

Exploration of Potential Tumor Markers for Lung Adenocarcinomas by Two‐Dimensional Gel Electrophoresis Coupled with Nano‐LC/MS/MS

To explore the potential tumor markers for lung adenocarcinoma, two‐dimensional gel electrophoresis (2DE) coupled with nano‐LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin‐1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S‐transferase Pi (GST‐Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up‐regulated in the cancer tissue. On the other hand, profilin was down‐regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.     

Direct Blue 71 staining of proteins bound to blotting membranes

A sensitive staining method for protein blots using Direct Blue 71 is described. It is based on the selective binding of dye molecules to proteins in acidic solution and produces bluish violet colored bands. It is a simple and rapid procedure, involving only staining and rinsing steps that occur within 7 min. The sensitivity of this method is 5-10 ng of protein on nitrocellulose (NC) and 10-20 ng on polyvinylidene difluoride (PVDF), which is tenfold better than that of the commonly used Ponceau S staining. Moreover, the staining is reversible for subsequent immunostaining, without impairing immunoreactivity. To remove the dye from the developed bands, changes in pH and hydrophobicity of the solvent are required. Due to its sensitivity, rapidity, simplicity, and low cost, this stain may be more practical than other dye-based stains or metal-based stains for routine laboratory purposes.     

A proteomic analysis of erythromycin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant human pathogen which is an important cause of pneumonia and bacteraemia. Over the past few years the incidence of antibiotic resistance among clinical isolates of S. pneumoniae has increased. Penicillin resistance is now widespread and the frequency of isolates that are resistant to erythromycin has risen. Erythromycin resistance in S. pneumoniae follows two basic patterns. The MLS erythromycin-resistant phenotype is due to the enzymatic methylation of ribosomal RNA that blocks erythromycin binding to the ribosome. Alternatively, in isolates of the M phenotype, a more recently documented mechanism, resistance is associated with an active efflux process that reduces intracellular levels of erythromycin. We used two-dimensional electrophoresis to examine the proteins synthesised by erythromycin-susceptible and -resistant S. pneumoniae. Erythromycin-resistant S. pneumoniae with the M phenotype showed a significantly increased synthesis of a 38,500 Dalton (pI 6.27) protein compared to susceptible isolates. Peptide mass mapping was used to identify the 38,500 Dalton protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was demonstrated that S. pneumoniae synthesised at least three forms of GAPDH that differed in their isoelectric points. The form of GAPDH possessing the most basic pI showed the increased synthesis in the erythromycin-resistant S. pneumoniae isolates. Alterations in the synthesis of GAPDH were only found for those erythromycin-resistant isolates possessing the M phenotype. S. pneumoniae isolates with the MLS phenotype were indistinguishable from the susceptible strains using the analytical conditions employed for the current study. The possible role of GAPDH in erythromycin resistance of S. pneumoniae is considered.     

The Antiproliferative and Apoptotic Effects of a Novel Quinazoline Carrying Substituted-Sulfonamides: In Vitro and Molecular Docking Study

In order to investigate for a new effective and safe anticancer drug, we synthesized a novel series of quinazoline containing biologically active substituted-sulfonamide moiety at 3- position 4a–n. The structure of the newly prepared compounds was proved by microanalysis, IR, ¹H-NMR, ¹³C-NMR and mass spectral data. All the synthesized compounds were evaluated for their in vitro cytotoxic activity in numerous cancer cell lines including A549, HepG-2, LoVo and MCF-7 and normal HUVEC cell line. The two most active compounds 4d and 4f were then tested for their apoptosis induction using DNA content and Annexin V-FITC/PI staining. Moreover, apoptosis initiation was also confirmed using RT-PCR and Western blot. To further understand the binding preferences of quinazoline sulfonamides, docking simulations were used. Among the fourteen new synthesized compounds, we found that compounds 4d and 4f exerted the strongest cytotoxicity against MCF-7 cells with an IC₅₀ value of 2.5 and 5 μM, respectively. Flow cytometry data revealed the ability of compounds 4d and 4f to mediate apoptosis and arrest cell cycle growth at G1 phase. Furthermore, RT-PCR and Western blot results suggested that both 4d and 4f activates apoptotic cell death pathway in MCF-7 cells. Molecular docking assessments indicated that compounds 4d and 4f fit perfectly into Bcl2’s active site. Based on the biological properties, we conclude that both compounds 4d and 4f could be used as a new type of anticancer agent, which provides a scientific basis for further research into the treatment of cancer.     

Simultaneous spectrophotometric determination of Tartrazine, Sunset Yellow and Ponceau 4R in commercial products by partial least squares and principal component regression multivariate calibration methods

Three multivariate calibration methods, partial least squares (PLS-1 and PLS-2) and principal component regression (PCR), were proposed and successfully applied to the simultaneous determination of three dyes, Tartrazine (E-102), Sunset Yellow (E-110) and Ponceau 4R (E-124) in mixtures by ultraviolet-visible absorption spectrophotometry. The designed and optimized training set of calibration was applied to the determination of the three dyes in several synthetic mixtures, containing 1.6–20.0 mg/L of Tartrazine, 3.2–40.0 mg/L of Sunset Yellow and 3.2– 36.0 mg/L of Ponceau 4R. 94.5–105.3% recovery values were obtained. Three commercial foods that contained the three dyes were also satisfactorily analyzed without separation step. The results obtained by the application of the three chemometric approaches in the commercial products are discussed and compared with those obtained by an HPLC method and very similar values were found by all methods. Repeatability and reproducibility studies (with the Student’s and F tests) were achieved over two series of nine standards for each dye, showing no significant differences at the 95% confidence level. Received: 27 October 1997 / Revised: 9 January 1998 / Accepted: 24 January 1998     

Bioinformatics analysis and verification of gene targets for renal clear cell carcinoma

BackgroundIt is estimated that there are 338,000 new renal-cell carcinoma releases every year in the world. Renal cell carcinoma (RCC) is a heterogeneous tumor, of which more than 70% is clear cell renal cell carcinoma (ccRCC). It is estimated that about 30% of new renal-cell carcinoma patients have metastases at the time of diagnosis. However, the pathogenesis of renal clear cell carcinoma has not been elucidated. Therefore, it is necessary to further study the pathogenesis of ccRCC.MethodsTwo expression profiling datasets (GSE68417, GSE71963) were downloaded from the GEO database. Differentially expressed genes (DEGs) between ccRCC and normal tissue samples were identified by GEO2R. Functional enrichment analysis was made by the DAVID tool. Protein-protein interaction (PPI) network was constructed. The hub genes were excavated. The clustering analysis of expression level of hub genes was performed by UCSC (University of California Santa Cruz) Xena database. The hub gene on overall survival rate (OS) in patients with ccRCC was performed by Kaplan-Meier Plotter. Finally, we used the ccRCC renal tissue samples to verify the hub genes.Results1182 common DEGs between the two datasets were identified. The results of GO and KEGG analysis revealed that variations in were predominantly enriched in intracellular signaling cascade, oxidation reduction, intrinsic to membrane, integral to membrane, nucleoside binding, purine nucleoside binding, pathways in cancer, focal adhesion, cell adhesion molecules. 10 hub genes ITGAX, CD86, LY86, TLR2, TYROBP, FCGR2A, FCGR2B, PTPRC, ITGB2, ITGAM were identified. FCGR2B and TYROBP were negatively correlated with the overall survival rate in patients with ccRCC (P < 0.05). RT-qPCR analysis showed that the relative expression levels of CD86, FCGR2A, FCGR2B, TYROBP, LY86, and TLR2 were significantly higher in ccRCC samples, compared with the adjacent renal tissue groups.ConclusionsIn summary, bioinformatics technology could be a useful tool to predict the progression of ccRCC. In addition, there are DEGs between ccRCC tumor tissue and normal renal tissue, and these DEGs might be considered as biomarkers for ccRCC.     

A new coumarin isolated from Sarcandra glabra as potential anti-inflammatory agent

One new coumarin, 3,5-dihydroxy-7-O-α-L-rhamno pyranosyl-2H-chromen-2-one (1), was isolated from the whole plant of Sarcandra glabra. The structure was elucidated by spectroscopic methods. Our results indicated that 1 significantly inhibit nitric oxide (NO) production in LPS-induced RAW264.7 macrophages. RT-PCR analysis indicated it inhibited iNOS mRNA expression. In addition, Western blot analysis showed that 1 attenuated LPS-induced synthesis of iNOS protein in the macrophages. These results suggest that 1 could be potential anti-inflammatory agent by down-regulating iNOS expression.