Simple Western: Bringing the Western Blot into the Twenty-First Century.

The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.

Translating Current Bioanalytical Techniques for Studying Corona Activity.

The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces.

EZH2 promotes tumor progression by increasing VEGF expression in clear cell renal cell carcinoma

Objectives. The present study is to evaluate the expression level of enhancer of zeste homolog 2 (EZH2) and vascular endothelial growth factor (VEGF), and analyze their correlations with clinicopathological characteristics and survival in patients with clear cell renal cell carcinoma (CCRCC). The effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line is investigated. Methods. The expression level of EZH2 and VEGF was detected in 185 primary CCRCC patients’ tissues using tissue microarray and immunohistochemistry. Small interfering RNA or enhanced green fluorescent protein transfection was employed to investigate the effect of EZH2 inhibition or overexpression on VEGF expression, apoptosis and cell proliferation in 786-O cells using flow cytometry, immunofluorescence microscopy, quantitative real-time reverse-transcription polymerase chain reaction and Western blot analysis. Results. High expression level of EZH2 and VEGF was observed in advanced CCRCC and correlated with the TNM stage (p = 0.013, p = 0.001) and distant metastasis (p = 0.011, p = 0.038), respectively. EZH2 was positively correlated with VEGF in CCRCC tissues (correlation coefficient = 0.850, p < 0.001). Kaplan–Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared to patients with negative EZH2 expression (34.3 vs. 67.2, p < 0.001). In 786-O cells, EZH2 silencing inhibited VEGF expression and cell proliferation while increasing apoptosis (p < 0.001). EZH2 overexpression promoted VEGF expression and cell proliferation while inhibiting apoptosis (p < 0.001). Conclusions. EZH2 correlates positively with VEGF and associates with adverse clinicopathologic characteristics and shorter survival time in CCRCC patients. EZH2 accelerates antiapoptosis and cell cycle in 786-O cells (AU)

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Expression of vimentin and survivin in clear cell renal cell carcinoma and correlation with p53

Purpose. This study focuses on investigating the expression correlation of vimentin, survivin and p53 in clear cell renal cell carcinoma (ccRCC) and the clinical significance. Methods. The mRNA and protein expression levels of the vimentin, survivin and p53 were determined in ccRCC and adjacent normal renal tissues, using quantitative real-time-polymerase chain reaction (qRT-PCR) and Western blot. We detected the expression and localization of vimentin, survivin and p53 protein in ccRCC by immunohistochemistrical SP method and analyzed the relationships among clinical pathologic parameters and patient prognosis. Results. The expression of vimentin and survivin was significantly increased in ccRCC compared with adjacent normal renal tissues, which were positively correlated with the pathological grade and clinical stage (P < 0.05). p53 was highly expressed in ccRCC compared with normal tissues (P < 0.05), which was not positively correlated with the pathological grade and clinical stage (P > 0.05). Furthermore, univariate and multivariate analysis showed that high expression levels of vimentin and survivin were independent prognostic indicators for ccRCC. The levels of vimentin and survivin were positively correlated in ccRCC (r = 0.428, P < 0.01). Conclusions. Reliable basis about biological behavior and prognosis judgments of ccRCC can be provided by combining detection of vimentin and survivin. Foundation and new ideas for gene therapy of ccRCC may be provided by further studying the relationship among vimentin, survivin and p53 in ccRCC (AU)

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Estrogen combined with progesterone decreases cell proliferation and inhibits the expression of Bcl-2 via microRNA let-7a and miR-34b in ovarian cancer cells

PURPOSE: This study evaluated the effect of estrogen (E2), progesterone (P4), and the combination of them (E2 + P4) on survival rate, apoptosis, and the expressions of Bcl-2, hsa-let-7a and has-miR-34b in primary ovarian cancer cells to provide new clues for the clinical treatments of ovarian cancer. METHODS: The primary ovarian cancer cells from 60 cases of clinical ovarian cancer tissues were isolated and then cultured. The survival rate of ovarian cancer cells after the treatment of E2, P4 and E2 + P4 was analyzed by MTT assay. Cell apoptosis rate and cell cycle were measured by FACS analysis. Moreover, the relative abundance of Bcl-2 and microRNAs (let-7a, miR-34b) expressions were detected by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS: Low concentrations of estrogen (10(-10), 10(-8), 10(-6 )mol/L) did not affect the proliferation of ovarian cancer cells. However, the high concentration of estrogen (10(-4 )mol/L) inhibited survival rate of ovarian cancer cells. Progesterone (10(-4 )mol/L) inhibited the proliferation of cancer cells. The combination of estrogen and progesterone significantly inhibited the survival rate of ovarian cancer cells with a time- and dose-dependent manner. High concentration of estrogen combined with progesterone (E2 + P4) induced apoptosis of ovarian cancer cells. E2 + P4 promoted the expression of let-7a and miR-34b and reduced the expression of Bcl-2 in ovarian cancer cells. When the expression of let-7a or/and miR-34b was inhibited using miRNA inhibitors, E2 + P4 treatment did not change the protein level of Bcl-2. CONCLUSION: E2 + P4 significantly inhibited the cell survival, promoted the cell apoptosis, induced the expression of let-7a and miR-34b, and reduced the expression of Bcl-2 in ovarian cancer cells (AU)

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The fastest Western in town: a contemporary twist on the classic Western blot analysis.

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

The increase in seroprevalence to Toxocara canis in asthmatic children is related to cross-reaction with Ascaris suum antigens

Background: It has been suggested that the presence of Toxocara canis larvae in lungs is an underlying factor in the onset of asthma. Although the association of asthma and seropositivity to Toxocara has been observed, there are no studies that indicate whether these antibodies are specific against T. canis antigens. Methods: Seroprevalence to T. canis excretion-secretion antigens (TcES Ag) were compared between asthmatic children (n=285) and non-asthmatic children (n=152), using IgG-ELISA and IgE-ELISA. The recognition patterns of TcES Ag were determined using Western blot (WB). Results: IgG-ELISA seroprevalence was 30.8% for asthmatic children and 19.7% for non-asthmatic children (p<0.05). IgE-ELISA seroprevalence was 7.7% for asthmatic children and 6.5% for non-asthmatic children, respectively (p>0.05). Sera of both groups positive to IgG-ELISA recognised 11 TcES Ag bands for IgG. No differences between the groups were observed regarding recognition patterns; the asthmatic group, however, presented significantly higher cross-reaction to Ascaris suum somatic antigens (AsS Ag) than the non-asthmatic group. Sixty-three sera from asthmatic children positive to IgG-ELISA were evaluated by WB for IgE and 58.7% revealed a recognition pattern for IgE. In the group of non-asthmatic children positive to IgG-ELISA, 80% presented IgE band recognition. No differences were observed between the groups regarding recognition patterns. Conclusions: The results observed suggest that differences in seroprevalence determined by IgG-ELISA between groups of asthmatic and non-asthmatic children reported by other authors occur because of a higher frequency of cross-reaction in asthmatic children (AU)

Investigating protein-protein interactions by far-Westerns.

The identification of protein interaction partners can often elucidate the function of the protein under investigation based on the "guilty by association" concept. Furthermore, the binding event between two proteins can be used as a functional assay when no such assay is available. Despite the large number of advanced techniques that are currently available for studying protein-protein interactions, far-Westerns or blot overlays are still very commonly used in the average laboratory setting due to their powerfulness. This is due to the simplicity and clarity in the results that they produce. Here, the details and mechanics of far-Westerns are discussed to help the reader choose amongst the different variations that exist depending on the question being investigated and the materials available to them. Some examples involving unique questions are also discussed in order to educate the reader on the versatility of far-Westerns. Finally, a troubleshooting section provides the reader with an understanding of the common problems that can be encountered and how these problems can be circumvented.

Western blot profile in HIV infection.

BACKGROUND: Although the overall sensitivity and specificity of the western blot (WB) test for detection of antibodies to various viral proteins is high, there has been a substantial difference in the timing of the appearance of antibody bands and their intensities during different stages of HIV infection. AIMS: Mapping different band patterns of Western blot results and correlating them with stages of HIV infection. METHODS: We performed a retrospective study with 1,467 HIV-1 infected cases confirmed by WB test between January 2002 to July 2005, with the objective of mapping different band patterns of western blot results and determining whether the presence or absence of certain bands was associated with any specific stage of HIV infection. For the interpretation of the WB results in this study, the guidelines recommended by NACO, India were followed. RESULTS: Reactivity with all the bands was the most commonly observed WB pattern, occurring in 92.91% (1363/1467) of cases, whereas the other 7.09% showed uncommon band patterns. Of all individual bands, p31 band was the most frequently missing one, absent in 7.09% cases. On classifying the WB reactive cases by the WHO clinical staging system, 38.45% (564/1467) were in Stage 1, 47.99% (704/1467) in stages 2 and 3 and 13.56% in stage 4. Correlation of CD4 cell counts with the various uncommon band patterns showed that only 5.56% (4/72) had counts in the 200-500 cells/microl range, whereas 45.83% and 48.61% had counts of < 200 and> 500 cells/microl respectively. CONCLUSION: Interpretation of the WB band pattern in combination with clinical features may be occasionally useful in predicting the stage of HIV infection.

Diferentes dosis de los fitoestrógenos genisteína y daidzeína afectan de distinto modo a la viabilidad celular y a la expresión de factores implicados en el desarrollo de cáncer de próstata / Different doses of genistein and daidzein phytoestrogens affect cell viability and expression of factors involved in the development of prostrate carcinoma differently

Introducción. Estudios epidemiológicos sugieren que los fitoestrógenos de la soja podrían ser responsables de la baja incidencia del carcinoma prostático (CaP) en poblaciones asiáticas. Las células de CaP expresan factores reguladores de la proliferación/viabilidad celular, así como factores capaces de interaccionar con las células en el microambiente óseo. En el presente estudio hemos evaluado los efectos de los fitoestrógenos de la soja, genisteína y daidzeína, sobre la viabilidad celular/apoptosis y sobre la expresión de la proteína relacionada con la parathormona (PTHrP) y su receptor (PTH1R), la osteoprotegerina (OPG) y el ligando del receptor activador del NF-kappa B (RANKL) en las células de CaP humano PC-3 y LNCaP. Pacientes y métodos. Las células, sembradas a 20.000 células/cm2 en RPMI con suero fetal bovino al 10%, se incubaron con distintas concentraciones de cada fitoestrógeno o el vehículo salino (control basal) durante 1-4 días. La viabilidad celular se evaluó por exclusión con azul de tripán y la apoptosis se determinó por citometría de flujo. La expresión génica de PTHrP se analizó por RT-PCR semicuantitativa con cebadores específicos. La expresión proteica de PTHrP, PTH1R, OPG y RANKL se determinó por transferencia western en extractos de proteína celular total o de membrana (RANKL). Resultados. Hemos encontrado que tanto la genisteína como la daidzeína en el rango µM disminuyen la viabilidad celular e incrementan la apoptosis; siendo la genisteína más potente y eficaz que la daidzeína en ambos tipos celulares. Además, estas isoflavonas a dosis ¾ nM aumentan la expresión de PTHrP y del PTH1R, así como la relación OPG/RANKL en estas células. Conclusiones. Estos hallazgos demuestran que diferentes dosis de genisteína y daidzeína inducen efectos diversos sobre las células de CaP que podrían afectar al desarrollo de este tumor(AU)

Introduction. Epidemiological studies suggest that soy phytoestrogens might be responsible for the low incidence of prostate carcinoma (PCa) in Asian populations. PCa cells express regulatory factors of cell proliferation and viability, and also several factors that interact with cells in the bone microenvironment. In the present study, we evaluated the effects of soy phytoestrogens genistein and daidzein on cell viability and apoptosis, and also on the expression of parathormone-related protein (PTHrP) and its receptor (PTH1R), osteoprotegerina (OPG) and receptor activator of nuclear factor kappa B ligand (RANK-L) in the human PCa cell lines PC-3 and LNCaP. Patients and methods. Cells were seeded at 20,000 cells/cm2 in RPMI with 10% fetal bovine serum, and then were incubated with different concentrations of each phytoestrogen or saline vehicle (basal control) for 1-4 days. Cell viability was evaluated by Trypan blue exclusion, and apoptosis was determined by flow citometry. Gene expression of PTHrP was analyzed by semiquantitative RT-PCR with specific primers. Protein expression of PTHrP, the PTH1R, OPG, and RANKL was determined by western blot in total cell protein or cell membrane (RANKL) extracts. Results. We found that both genistein and daidzein, at µM range, decrease cell viability and increase apoptosis; but genistein was more potent and efficient than daidzein in both PCa cell lines. In addition, these isoflavones, at ¾ nM, increase the expression of PTHrP and the PTH1R, and also the OPG/RANKL ratio in these cells. Conclusions. These findings demonstrate that different concentrations of genistein and daidzein induce distinct effects on PCa cells that might affect PCa development(AU)

Detection of genetically modified organisms in foods.

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.

Human T-lymphotropic virus type I/II. Status of enzyme immunoassay and western blot testing in the United States in 1989 and 1990.

In three performance evaluation surveys, panels that consisted of human T-lymphotropic virus type I or type II (HTLV-I/II) antibody-positive and -negative plasma samples were mailed to laboratories that voluntarily participated in the Centers for Disease Control Model Performance Evaluation Program. Donor samples were identical among surveys. In each survey, more than 98% of the laboratories reported enzyme immunoassay (EIA) test results; about 11% also reported results of Western blot (WB) testing. Variation in analytic sensitivity (96.7% to 99.4%) and specificity (98.3% to 99.5%) of EIA tests was noted in the three surveys. For WB testing, no nonreactive interpretations were reported for HTLV-I/II antibody-positive samples in any survey; however, indeterminate interpretations were reported for 35.2% to 40.7% of the WB tests that were performed on HTLV-I/II antibody-positive samples. More than 95% of these indeterminate WB test interpretations were reported for HTLV-II antibody-positive samples. Although HTLV-I/II antibody tests are generally sensitive and specific, their accuracy could be further improved by increasing the specificity of EIA tests and the sensitivity of WB tests.