RESUMO
We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10-50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.
Assuntos
Eletroforese em Gel de Poliacrilamida/normas , Escherichia coli/química , Plasmídeos , Clonagem Molecular , Escherichia coli/genética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Padrões de ReferênciaRESUMO
Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.
Assuntos
Western Blotting/normas , Eletroforese em Gel de Poliacrilamida/normas , Miocárdio/metabolismo , Proteínas/análise , Animais , Calibragem , Camundongos , Padrões de ReferênciaRESUMO
INTRODUCTION: The quantitative determination of urinary Cystatin C (cyst-C) associated with the qualitative analysis of its polymorphisms is an excellent method for early identification of newborns predisposed to renal function impairment. PETIA, PENIA and EIA are the immunometric methods used for the quantitative determination of cyst-C in human biologic fluid but they have limitations and do not allow qualitative analysis. The present study is a validation of Immunoblot SDS-PAGE for the qualitative and quantitative analysis of urinary cyst-C. METHODS: Urine was collected from neonates in the nursey at S. Maria della Misericordia Hospital. Urinary cyst-C was investigated by the immunoblot SDS-PAGE and by reading of optical density. RESULTS: The qualitative analysis showed two different molecular forms: a reactivity at about 70 KDa in all samples and a reactivity at 13 KDa in a limited number of samples. This analysis allows the correlation of the polymorphisms of cyst-C with specific alterations of renal function in newborns. The quantitative analysis is specific, sensitive and accurate. In fact the coefficient of variation for assay precision was 10% and for assay accuracy was ±10%, the detection limit was 0.009 ng/ µL and the calibration line has satisfactory linearity (range 0.02-0.3 ng/ µL). The stability of urinary cyst-C was acceptable, even without the use of protease inhibitors, as long as the assay was performed on freshly recruited urine or immediately after thawing the samples, which had been stored for up to six months. CONCLUSION: Immunoblot SDS-PAGE analysis is a valid method of obtaining a qualitative and quantitative analysis of urinary cyst-C. This method presents unique information about a previously unknown 70 KDa cyst-C form. The assay may offer potential diagnostic information not available with immunometric method.
Assuntos
Bioensaio/métodos , Biomarcadores/urina , Western Blotting/métodos , Cistatina C/urina , Eletroforese em Gel de Poliacrilamida/métodos , Nefropatias/urina , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Humanos , Recém-Nascido , Nefropatias/diagnóstico , Espectrometria de Massas em Tandem/métodosRESUMO
Nonerythropoietic erythropoietins (EPOs) are investigated for their high antioxidant properties. A new drug candidate under clinical investigation to treat brain diseases is Neuro-EPO, produced by selecting EPO isoforms with low sialic acid content. Intranasal administration allows to bypass the blood-brain barrier to get a fast and concentrated delivery to the brain. The aims of this project were to characterize Neuro-EPO with anti-doping methods used to detect conventional recombinant EPOs (isoelectric focusing [IEF] and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and to evaluate the window of detection of Neuro-EPO in brain and blood (plasma) after a single intranasal administration in rats. Neuro-EPO drug analyzed by IEF-PAGE presented a very basic profile completely detected only when using a 2-8 or 2-10 pH gradient instead of the conventional 2-6 pH gradient. Its profile consisted in six main bands that did not interfere with endogenous EPO profile from human or rat. After SDS-PAGE, a broad band was detected for Neuro-EPO in the same area as endogenous EPO, making Neuro-EPO identification very difficult by this approach. Therefore, IEF was the method for identification chosen after administration in rats. Neuro-EPO was clearly identified in blood 2 and 6 h after the delivery. Fainter signals were obtained between 12 and 48 h, but some characteristic very basic bands remained detectable. Surprisingly, brain extracts did not show the presence of Neuro-EPO even 2 h after administration, indicating a fast degradation or elimination from the brain to the bloodstream. This experiment indicated that detection of Neuro-EPO after intranasal delivery should be possible for a few days.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/administração & dosagem , Eritropoetina/sangue , Detecção do Abuso de Substâncias/métodos , Administração Intranasal , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Eletroforese em Gel de Poliacrilamida/normas , Masculino , Ratos , Ratos Wistar , Detecção do Abuso de Substâncias/normasRESUMO
Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
Assuntos
Leucócitos Mononucleares/química , Proteoma/isolamento & purificação , Proteômica/métodos , Software , Sequência de Aminoácidos , Cromatografia Líquida/normas , Misturas Complexas/química , Eletroforese em Gel de Poliacrilamida/normas , Análise de Fourier , Humanos , Flebotomia/normas , Proteômica/normas , Espectrometria de Massas em Tandem/normasRESUMO
PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log10 (dsDNA length in bp) = 1.66 × log10 (polyP length in phosphate units) - 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE.
Assuntos
DNA/química , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Polifosfatos/análise , Polifosfatos/química , DNA/análise , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Virtual electrophoresis in proteomics can be used to search localization of proteins and their proteoforms (especially those existing in low concentrations), to identify proteoforms found in experiments etc. Although the problem of predicting the isoelectric point is well studied, the need of electrophoretic shift correction is usually ignored. Researchers simply use the brutto molecular weight of the protein. In this study four data sets taken from the literature sources and the SWISS-2DPAGE database have been used to build correction equations for prediction of the electrophoretic shift (123, 72, 118 and 470 points, respectively). Two groups of models were built. The first model was based on the amino acid composition of proteins, the second one, on analysis of parameters calculated by amino acid sequences (theoretical molecular weight, hydrophobicity, charge distribution, ability to form helix structures). The coefficient of determination ranged from 0.35 to 0.75 in each single set, but cross-prediction between samples did not gave satisfactory results. At the same time, the direction of correction was predicted correctly in 74% of cases. After combining of the samples and dividing pooled data into 2 representative sets, the coefficient of determination during in the process of learning ranged from 0.44 to 0.51, and R2 of predictions were not less than 0.39. The direction of correction was predicted correctly in 80% of cases. This prediction models have been integrated into the program pIPredict v.2, freely available at http://www.ibmc.msk.ru/LPCIT/pIPredict.
Assuntos
Aminoácidos/química , Eletroforese em Gel Bidimensional/estatística & dados numéricos , Eletroforese em Gel de Poliacrilamida/estatística & dados numéricos , Modelos Estatísticos , Proteínas/isolamento & purificação , Interface Usuário-Computador , Sequência de Aminoácidos , Bases de Dados Factuais , Eletroforese em Gel Bidimensional/normas , Eletroforese em Gel de Poliacrilamida/normas , Interações Hidrofóbicas e Hidrofílicas , Internet , Ponto Isoelétrico , Peso Molecular , Conformação Proteica em alfa-Hélice , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Proteínas/química , Eletricidade EstáticaRESUMO
Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.
Assuntos
Eletroforese em Gel de Poliacrilamida/normas , Fibrossarcoma/enzimologia , Gelatina/metabolismo , Gelatinases/metabolismo , Metaloproteinases da Matriz/análise , Etilenocloroidrina/análogos & derivados , Etilenocloroidrina/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Células Tumorais CultivadasRESUMO
Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were <10%, percentage recovery was between 97% and 109% with a high linearity (r = 0.99). HRE allowed the visualisation of a faint band of albumin and a diffused band between alpha and beta zones in healthy cats, while profiles from diseased cats were variable. Albumin (mg/dl) and urine albumin:creatinine ratio (UAC) were significantly ( P <0.05) different between healthy and diseased cats. After ROC analysis, UAC values of 0.035 and 0.074 had a high sensitivity and high specificity, respectively, to classify proteinuria and identify borderline proteinuric cats. Moreover, a UAC of 0.017 had a high sensitivity in distinguishing between healthy and diseased cats. However, UAC was not able to distinguish between renal (CKD) and non-renal diseases (LUTD/OTHER), probably owing to the pathophysiology of CKD in cats, which is characterised by low-grade proteinuria and less glomerular involvement than in dogs. Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.
Assuntos
Albuminúria/veterinária , Doenças do Gato/diagnóstico , Eletroforese em Gel de Poliacrilamida/veterinária , Insuficiência Renal Crônica/veterinária , Urinálise/veterinária , Albuminúria/diagnóstico , Animais , Doenças do Gato/urina , Gatos , Creatinina/urina , Eletroforese em Gel de Poliacrilamida/normas , Feminino , Masculino , Insuficiência Renal Crônica/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Oxidative stress has been extensively studied due to its correlation with cellular disorders and aging. In proteins, one biomarker of oxidative stress is the presence of carbonyl groups, such as aldehyde and ketone, in specific amino acid side chains such as lysine, proline, arginine and threonine, so-called protein carbonylation (PC). PC study is now a growing field in general and medical science since PC accumulation is associated with various pathologies and disorders. At present, enzyme-linked immunosorbent assays (ELISA) seem to be the most robust method of quantifying the presence of carbonyl groups in proteins, despite having some recognised caveats. In parallel, gel-based approaches present cross-comparison difficulties, along with other technical problems. As generic PC analyses still suffer from poor homogeneity, leading to cross-data analysis difficulties and poor results overlap, the need for harmonisation in the field of carbonyl detection is now widely accepted. This study aims to highlight some of the technical challenges in proteomic gel-based multiplexing experiments when dealing with PC in difficult samples like those from Caenorhabditis elegans, from protein extraction to carbonyl detection. We demonstrate that some critical technical parameters, such as labelling time, probe concentration, and total and carbonylated protein recovery rates, should be re-addressed in a sample-specific way. We also defined a procedure to cost-effectively adapt CyDye™-hydrazide-based protocols to specific samples, especially when the experimental interest is focused on studying differences between stimulating conditions with a maximised signal-to-noise ratio. Moreover, we have improved an already-existing powerful solubilisation buffer, making it potentially useful for hard-to-solubilise protein pellets. Lastly, the depicted methodology exemplifies a simple way of normalising carbonyl-related signal to total protein in SDS-PAGE multiplexing experiments. Within that scope, we also proposed a simple way to quantify carbonyl groups by on-gel spotting diluted dye-containing labelling buffer. Proof of the robustness of the procedure was also highlighted by the high linear correlation between the level of carbonyls and the ultraviolet exposure duration of whole worms (R2=0.993). Altogether, these results will help to standardise existing protocols in the growing field of proteomic carbonylation studies.
Assuntos
Envelhecimento/metabolismo , Benchmarking , Proteínas de Caenorhabditis elegans/isolamento & purificação , Caenorhabditis elegans/metabolismo , Carbonilação Proteica , Proteômica/normas , Envelhecimento/genética , Animais , Biomarcadores/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes/química , Estresse Oxidativo , Proteômica/métodos , Razão Sinal-Ruído , Coloração e Rotulagem/métodos , Coloração e Rotulagem/normasRESUMO
EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.
Assuntos
Dopagem Esportivo , Eletroforese em Gel de Poliacrilamida , Eritropoetina/sangue , Fragmentos Fc das Imunoglobulinas/sangue , Focalização Isoelétrica , Substâncias para Melhoria do Desempenho/sangue , Proteínas Recombinantes de Fusão/sangue , Detecção do Abuso de Substâncias/métodos , Western Blotting , Calibragem , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida/normas , Humanos , Focalização Isoelétrica/normas , Ponto Isoelétrico , Medições Luminescentes , Peptídeo Hidrolases/metabolismo , Valor Preditivo dos Testes , Proteólise , Padrões de Referência , Reprodutibilidade dos Testes , Detecção do Abuso de Substâncias/normas , Espectrometria de Massas em TandemRESUMO
Quantitative analysis of electrophoresis gels is an important part in molecular cloning, as well as in protein expression and purification. Parallel quantifications in yield and purity can be most conveniently obtained from densitometric analysis. This communication reports a comprehensive, reliable and simple protocol for gel quantification and documentation, applicable for single samples and with special features for protein expression screens. As major component of the protocol, the fully annotated code of a proprietary open source computer program for semi-automatic densitometric quantification of digitized electrophoresis gels is disclosed. The program ("GelQuant") is implemented for the C-based macro-language of the widespread integrated development environment of IGOR Pro.
Assuntos
Eletroforese em Gel de Poliacrilamida/normas , Fragmentos de Peptídeos/isolamento & purificação , Príons/isolamento & purificação , Software , Coloração e Rotulagem/métodos , Animais , Calibragem , Clonagem Molecular , Densitometria/instrumentação , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Camundongos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Plasmídeos/química , Plasmídeos/metabolismo , Príons/biossíntese , Príons/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Padrões de ReferênciaRESUMO
Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.
Assuntos
Anticorpos Monoclonais Humanizados/análise , Anticorpos Monoclonais/análise , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel Bidimensional/normas , Eletroforese em Gel de Poliacrilamida/normas , Glicosilação , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , Isoformas de Proteínas/análise , Estabilidade Proteica , Controle de Qualidade , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Temperatura , TrastuzumabRESUMO
BACKGROUND: The determination of matrix metalloproteases (MMPs) is relevant in many pathophysiological conditions, especially if associated with extracellular matrix remodeling; however, the results obtained are closely linked to the method used and are not directly comparable. The aim of this study was to perform a reappraisal of quantitative gel zymography technique for MMPs in human plasma, to use for comparison with commercially available ELISA and in those experimental conditions where the MMP active form needs to be revealed. METHODS: The critical methodological parameters of zymography were checked and a comparison with a routinely used ELISA was performed. RESULTS: Sensitivity and reproducibility levels of zymography are suitable for detection of MMP-9 in human plasma, providing results closely related to those obtained by ELISA. CONCLUSIONS: Analytical parameters of zymography were suitable for detection of MMPs in human plasma. Quantitative zymography for MMPs is an alternative method for comparing the results of ELISA widely employed for MMP determination, thus reducing the discrepancies between laboratories regarding gelatinase assay.
Assuntos
Eletroforese em Gel de Poliacrilamida/normas , Ensaios Enzimáticos/normas , Metaloproteinase 9 da Matriz/sangue , Análise Química do Sangue/normas , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Modelos Logísticos , Masculino , Metaloproteinases da Matriz/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals.
Assuntos
Coloração pela Prata/instrumentação , Resinas Acrílicas/química , DNA/química , Eletroforese em Gel de Poliacrilamida/economia , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Indicadores e Reagentes , Proteínas/química , Padrões de Referência , Coloração pela Prata/economia , Coloração pela Prata/métodosRESUMO
Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, ß-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix.
Assuntos
Bile/metabolismo , Ciclídeos/metabolismo , Monitoramento Ambiental/métodos , Fígado/metabolismo , Metalotioneína/isolamento & purificação , Animais , Bile/química , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Monitoramento Ambiental/normas , Fígado/química , Metalotioneína/metabolismo , Espectrofotometria/métodos , Espectrofotometria/normas , Poluentes Químicos da Água/metabolismoRESUMO
Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [(3)H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin-solubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT(3A) receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.
Assuntos
Membrana Celular/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Animais , Transporte Biológico , Células CHO , Calibragem , Cricetinae , Eletroforese em Gel de Poliacrilamida/normas , Feminino , Ácido Fólico/metabolismo , Glicosilação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Peso Molecular , Oócitos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Transportador de Folato Acoplado a Próton/química , Padrões de Referência , Xenopus laevisRESUMO
Over the last decade, science has greatly improved in the area of protein sizing and characterization. Efficient high-throughput methods are now available to substitute for the traditional labor-intensive SDS-PAGE methods, which alternatively take days to analyze a very limited number of samples. Currently, PerkinElmer(®) (Caliper) has designed an automated chip-based fluorescence detection method capable of analyzing proteins in minutes with sensitivity similar to standard SDS-PAGE. Here, we describe the use and implementation of this technology to characterize and screen a large number of formulations of target glycoproteins in the 14-200 kDa molecular weight range.
Assuntos
Eritropoetina/química , Fragmentos Fc das Imunoglobulinas/química , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Glicoproteínas/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Análise Serial de Proteínas/métodos , Análise Serial de Proteínas/normas , Proteínas Recombinantes de Fusão/química , Padrões de Referência , Espectrometria de FluorescênciaAssuntos
Eletroforese em Gel de Poliacrilamida , Lipoproteínas LDL/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Povo Asiático , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/normas , Feminino , Humanos , Lipoproteínas LDL/classificação , Masculino , Pessoa de Meia-Idade , Valores de Referência , República da Coreia , Adulto JovemRESUMO
In this study, a Veterinary Department repository composed by paired formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FrFr) sets of the same tissues, routinely archived in the typical conditions of a clinical setting, was exploited to perform a comparative evaluation of the results generated by GeLC-MS/MS (1-DE followed by in-gel digestion and LC-MS/MS) and spectral counting with the two types of archival samples. Therefore, two parallel differential proteomic studies were performed using 3 canine mammary carcinomas and 3 normal controls in a paired fashion (6 FrFr and 6 FFPE in total). As a result, the FrFr and FFPE differential proteomic datasets exhibited fair consistency in differential expression trends, according to protein molecular function, cellular localization, networks, and pathways. However, FFPE samples were globally slightly less informative, especially concerning the high-MW subproteome. As a further investigation, new insights into the molecular aspects of protein fixation and retrieval were obtained. In conclusion, archival FFPE samples can be reliably used for differential proteomics studies employing a spectral counting GeLC-MS/MS approach, although some typical biases need to be taken into account, and FrFr specimens (when available) should still be considered as the gold standard for clinical proteomics.