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1.
Food Chem ; 399: 133966, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36007444

RESUMO

Tropomyosin, a myofibrillar muscle protein, has been recognized as a finfish allergen. In this study, tropomyosin from Atlantic cod fillets (Gadus morhua, CTM) was purified using a two-step purification strategy (isoelectric precipitation and anion-exchange chromatography). CTM structural configuration in two sample matrices (impure and pure) were elaborated using different polyacrylamide gel electrophoresis (native, non-reducing, and reducing PAGE). Their corresponding immunoblots were conducted to investigate CTM antigenicity under three conditions. Overall, CTM retained solubility, integrity, and antigenicity after heat treatment. Three CTM monomeric α-type isoforms (33 kDa) were identified using two-dimensional PAGE. Under native condition, the vast majority of CTM existed in the disulfide-reduced dimeric form (66 kDa). Under non-reducing condition, sodium dodecyl sulfate (anionic surfactant) broke CTM dimers, leaving monomers and disulfide-induced tetramers. Under reducing condition, ß-mercaptoethanol (thiol reducing agent) dissociated disulfide-linked CTM tetramers (132 kDa) into monomers (33 kDa). CTM retained antigenicity regardless of structural configuration under different conditions.


Assuntos
Gadus morhua , Animais , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Peixes , Gadus morhua/metabolismo , Tropomiosina/metabolismo
2.
J Chromatogr A ; 1679: 463389, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35933772

RESUMO

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 - 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, ß-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.


Assuntos
Actinas , Eletroforese Capilar , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Dodecilsulfato de Sódio
3.
Anal Biochem ; 655: 114833, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35961398

RESUMO

This manuscript describes the formation of an artifact shoulder peak with a slightly larger retention time than the main peak under the standard non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS) analysis of a therapeutic recombinant protein X, and clarifies the formation mechanism of the artifact caused by N-ethylmaleimide (NEM) during the sample preparation procedure. A design of experiment (DoE) approach was used to investigate the impact of the factors on the formation of the impurity. Additionally, orthogonal analytical experiments were performed to study the root cause of this phenomenon. The results consistently suggested that the Michael addition reaction between NEM and lysine residues in protein X, and decreased electrophoretic mobility due to increased molecular weight, was the root cause for the artifact, which could be partially inhibited by modifications of incubation conditions. Thus, before performing the nrCE-SDS method, the effects of alkylation reagents and sample preparation procedure on analytical results need to be considered seriously.


Assuntos
Artefatos , Eletroforese Capilar , Alquilação , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida , Etilmaleimida , Indicadores e Reagentes , Dodecilsulfato de Sódio/química
4.
Vet Parasitol ; 310: 109774, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35981467

RESUMO

The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.


Assuntos
Doenças do Gato , Dicrocoeliidae , Infecções por Trematódeos , Animais , Doenças do Gato/diagnóstico , Gatos , Cromatografia Líquida/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Óvulo , Espectrometria de Massas em Tandem/veterinária , Infecções por Trematódeos/veterinária
5.
Anal Biochem ; 654: 114817, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35863464

RESUMO

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.


Assuntos
Corantes Fluorescentes , Soroalbumina Bovina , Trifosfato de Adenosina , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Géis , Sefarose , Dodecilsulfato de Sódio , Coloração e Rotulagem
6.
Microb Biotechnol ; 15(9): 2337-2350, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35849816

RESUMO

Pseudomonas sp. strain 166 was isolated from soil samples from Changbai Mountains. A novel bacteriocin PA166 from Pseudomonas sp. 166 was purified using ammonium sulfate, dextran gel chromatography column and Q-Sepharose column chromatography successively. The molecular mass of bacteriocin PA166 was found to be 49.38 kDa by SDS-PAGE and liquid chromatography-mass spectrometry (MS)/MS. Bacteriocin PA166 showed stability at a wide range of pH (2-10), and thermal stability (40, 60, 80 and 100°C). The bacteriocin PA166 antimicrobial activity was slightly inhibited by Ca2+ , K+ and Mg2+ . The minimum bactericidal concentrations of bacteriocin PA166 against five Pasteurella multocida strains ranged from 2 to 8 µg ml-1 . Bacteriocin PA166 showed low cytotoxicity and a higher treatment index (TI = 82.51). Fluorescence spectroscopy indicated that bacteriocin PA166 destroyed the cell membrane to exert antimicrobial activity. In summary, bacteriocin PA166 had strong antibacterial activity, high TI and low toxicity, and hence could serve as a potential clinical therapeutic drug.


Assuntos
Bacteriocinas , Antibacterianos/química , Bacteriocinas/farmacologia , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Pseudomonas
7.
Arch Razi Inst ; 77(1): 23-28, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35891759

RESUMO

Scant information is available on the immunological aspect of Linguatula serrata causing linguatulosis in humans and animals. The present study aimed to analyze the content of crude somatic extracts and excretory-secretory products of L. serrata nymphs to detect the immune response of sheep and immunogenic proteins of the parasite. After collecting the nymphs, somatic extracts were prepared by sonication. Excretory secretory products were prepared by the incubation of nymphs in RPMI medium at 37°C with 5% CO2. Somatic and excretory-secretory proteins were isolated using SDS-PAGE. The immunogenic properties of the resulting proteins were determined using immunoblotting and positive sera from sheep infected with visceral linguatulosis. The total content of somatic extracts and excretory-secretory products of L. serrata nymphs analyzed by SDS-PAGE (12% gel) revealed two protein patterns with more than 18 and 9 strong bands, respectively. Immunoblots using sera samples of sheep infected with the parasite, somatic extracts and excretory-secretory products demonstrated 12 and 3 antigenic proteins with molecular weights mostly in the range of 24-100 kDa and an antigen more than 180 kDa. Three common immunodominant antigenic proteins with molecular weights of 38 and 57, as well as an antigen of more than 180 kDa, were detected in the somatic extracts and excretory-secretory products of L. serrata nymphs in sheep with visceral linguatulosis. These antigens can be considetered prime candidates for future serodiagnosis and immunoprotective studies of the parasite.


Assuntos
Doenças Parasitárias em Animais , Pentastomídeos , Doenças dos Ovinos , Animais , Eletroforese em Gel de Poliacrilamida/veterinária , Ninfa/fisiologia , Doenças Parasitárias em Animais/parasitologia , Pentastomídeos/fisiologia , Ovinos , Doenças dos Ovinos/parasitologia
8.
J Pharm Biomed Anal ; 219: 114926, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-35839578

RESUMO

Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.


Assuntos
Detergentes , Proteínas de Membrana , Animais , Detergentes/química , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mamíferos , Camundongos , Octoxinol
9.
Protein J ; 41(4-5): 438-443, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35895218

RESUMO

A new method for photosensitized polymerization of polyacrylamide gels was proposed. Photopolymerization of acrylamide/N,N'-methylenebisacrylamide (AM/Bis) was assisted with combination of catalyst ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) and photoinitiator riboflavin (RF). The prepared cross-linked AM/Bis + EDTA/RF gels were tested in electrophoretic SDS-PAGE system at high concentration of AM (20 wt%). The efficiency of these systems for electrophoretic separation of histones of human blood lymphocytes was demonstrated. In principle, such gels with small pores in the separation zone can offer advantages for resolution of proteins. The advantages of proposed method also include simple technique and possibility of gel preparation in a timely manner (for 10-15 min). However, in microporous gel systems some limitations in electroblotting technique could occur, which is particularly crucial for hydrophobic proteins.


Assuntos
Proteínas , Riboflavina , Ácido Edético , Eletroforese em Gel de Poliacrilamida , Géis , Humanos , Proteínas/química
10.
J Agric Food Chem ; 70(23): 7211-7219, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35666675

RESUMO

High-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) in a mature grain play important roles in the formation of a glutenin macropolymer and gluten quality. To characterize the expressed glutenin genes of the bread wheat variety Xinmai 26 during seed development, a total of 18 full-length transcripts were obtained by the newly emerged third-generation RNA sequencing of the PacBio Sequel II platform, including 5 transcripts of HMW-GS genes and 13 transcripts of LMW-GS genes (8 intact genes and 5 pseudogenes). Combined with the patterns of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), allelic types of the obtained glutenin genes were, respectively, determined, wherein molecular characterization deduced by transcript1528 (1Dx5) and transcript907 (Glu-A3c) indicated their great influence on dough quality. In addition, a specific functional marker dCAPS5 was developed for the single-nucleotide substitution at position 353 of the 1Dx5 subunit, which was further intensively compared with the other proposed markers to efficiently utilize the 1Dx5 subunit with the extra cysteine residue. This study provides an efficient method to accurately identify and utilize glutenin genes in bread wheat, which is helpful in understanding the contributions of glutenin genes to wheat quality.


Assuntos
Pão , Triticum , Pão/análise , Cisteína/genética , Eletroforese em Gel de Poliacrilamida , Glutens/química , Glutens/genética , Peso Molecular , Análise de Sequência de RNA , Triticum/química , Triticum/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-35700648

RESUMO

PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.


Assuntos
Heparina , Muramidase , Antivirais , Cromatografia , Eletroforese em Gel de Poliacrilamida , Muramidase/química , Polietilenoglicóis/química
12.
Artigo em Inglês | MEDLINE | ID: mdl-35644319

RESUMO

Lectins are carbohydrate-binding proteins that possess specific sugar-binding properties and are involved in various biological activities in different organisms. In this study, purification, characterization, and cDNA cloning of a brittle star lectin, designated as Ophioplocus japonicus agglutinin (OJA), were conducted. OJA was isolated from the brittle star O. japonicus by affinity chromatography on a Sephadex G-25 column, followed by ion-exchange chromatography on a Resource Q column. This lectin yielded distinct bands at approximately 176 or 17 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing or reducing conditions, respectively. It also exhibited Ca2+-dependent hemagglutination activity, which, however, was not affected by other metal cations, such as Ba2+, Co2+, Cu2+, Zn2+, Fe2+, Mg2+, and Mn2+. The OJA activity was strongly inhibited by glucose and xylose among the monosaccharides tested, and by bovine thyroglobulin among the glycoproteins tested. Cloning of the OJA cDNA revealed that its primary structure contained the C-type lectin domain (CTLD). The results of this study showed that OJA is an echinoderm-derived glucose/xylose-specific lectin that belongs to the C-type lectin superfamily.


Assuntos
Lectinas Tipo C , Xilose , Animais , Bovinos , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Glucose , Peso Molecular
13.
Anal Biochem ; 653: 114788, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35732212

RESUMO

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV-Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.


Assuntos
Hemeproteínas , Proteínas de Transporte , Eletroforese em Gel de Poliacrilamida , Heme , Espectrometria de Massas/métodos
14.
Anal Biochem ; 653: 114789, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35738440

RESUMO

Tris-Glycine-SDS is the most commonly used running buffer for SDS-PAGE. Relatively long running times, poor resolution of small molecular weight proteins and excessive heat at higher voltages impede its utility for high throughput downstream applications such as western blot. Here we describe a protocol for gradient-like simultaneous separation of small (<10 kDa) and large (>400 kDa) proteins in a single percentage polyacrylamide Tris-Acetate gel using a novel running buffer composed of Tris, Tricine and HEPES.


Assuntos
Proteínas , Corrida , Eletroforese em Gel de Poliacrilamida , Glicina/análogos & derivados , HEPES , Peso Molecular
15.
Sci Rep ; 12(1): 9434, 2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35676391

RESUMO

The present study aims to assess the effects of thermal and chemical inactivating procedures, that can be used for SARS-CoV-2 inactivation, on different salivary analytes. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile and a panel of 25 specific biomarkers of oxidative status, stress, metabolism and tissue damage were evaluated in samples subjected to different treatments: thermal (65 °C or 92 °C) and chemical with detergents [sodium dodecyl sulphate (SDS), Triton X-100 or NP-40]. Salivary SDS-PAGE profile was most affected by heating at 92 °C, with three and two protein bands decreasing and increasing their expression levels, respectively. This treatment also affected the results of several enzymes, with some of them being also affected by heating at 65 °C and incubation with SDS. The use of Triton X-100 or NP-40 resulted in increased values of cortisol, triglycerides and glucose, not affecting the other tested biomarkers. The present results will help researchers and clinicians to select the best protocols to work in safe conditions with saliva, taking into account the target analyte planned to be measured.


Assuntos
COVID-19 , Saliva , Eletroforese em Gel de Poliacrilamida , Humanos , Octoxinol/farmacologia , Proteínas , SARS-CoV-2
16.
Food Chem ; 393: 133331, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35661606

RESUMO

Understanding wheat gliadin-proanthocyanidin (PA) interactions would be useful to systematically control foams and gels, create novel textures, and reduce inflammatory reactions. This work aimed to determine the effects of heat (50-90 °C) on gliadin-proanthocyanidin (PA) interactions. Gliadin-PA mixtures were heated for 30 min in aqueous ethanol, and resulting morphology, fluorescence, and MW distribution were analyzed. Atomic force microscopy showed that PA greatly increased gliadin particle size, especially with heat. PA significantly quenched gliadin's tryptophan fluorescence. Further fluorescence data analysis indicated that PA interacted with gliadins through static quenching, primarily via hydrophobic interactions, and that 75 °C treatment yielded the greatest gliadin-PA interactions, likely because the proteins unraveled and exposed residues for interaction. PA appeared to interact mostly with ω-gliadins, based on their absence in the SDS-PAGE gel. Though it has been overshadowed in previous studies by non-covalent interactions, staining of quinoproteins indicated that PA covalently cross-linked gliadins at pH âˆ¼ 6.


Assuntos
Gliadina , Proantocianidinas , Eletroforese em Gel de Poliacrilamida , Gliadina/química , Temperatura Alta , Triticum/química
17.
Arch Biochem Biophys ; 726: 109241, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35667908

RESUMO

A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin : myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.


Assuntos
Miofibrilas , Tropomiosina , Actinas/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Coelhos , Tropomiosina/metabolismo , Troponina C/metabolismo
18.
Methods Mol Biol ; 2497: 107-115, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35771438

RESUMO

The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.


Assuntos
Membranas Mitocondriais , Fosforilação Oxidativa , Animais , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Camundongos , Eletroforese em Gel de Poliacrilamida Nativa/métodos
19.
Methods Mol Biol ; 2497: 339-348, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35771456

RESUMO

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.


Assuntos
Fibroblastos , Membranas Mitocondriais , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Humanos , Eletroforese em Gel de Poliacrilamida Nativa/métodos
20.
Anal Biochem ; 652: 114751, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35667451

RESUMO

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a standard method for protein analysis. However, the stacking gel employed for the standard SDS-PAGE is transparent and indistinct. Therefore, loading samples into stacking gel wells can be challenging. Accordingly, an acidic dye (tartrazine, brilliant blue FCF, or new coccine), which allowed easy visualization of the stacking gel wells and straightforward sample loading into these wells, was added to the SDS-PAGE stacking gel to resolve this issue. Moreover, the performance of these gels was comparable to that of non-colored standard gels. Thus, researchers worldwide could adopt this simple method for protein analysis.


Assuntos
Proteínas , Western Blotting , Eletroforese em Gel de Poliacrilamida , Géis , Proteínas/análise , Dodecilsulfato de Sódio
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