This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

Protokolü kurumların insan araştırma etik kurulunun yönergeleri takip eder. Sağlıklı insan donörlerden Buffy coat Bölge Kan Transfüzyon Merkezi (Lille, Fransa) elde edilmiştir. Buffy kat elde edilen Numune bir Inserm koleksiyon (n ° DC2010-1209) olarak ilan edilmiştir. 1. Malzeme ve Kültür Medya Hazırlık <ol><li> 1x PBS elde etmek için steril damıtılmış su içinde, 10X fosfat tamponlu salin (PBS) ile seyreltilir. </li><li> Ve% 10 toplanmış insan serumu olmadan gentamisin (40 ug / ml) ve L-glutamin (2 mM) ile takviye edilmiş RPMI 1640 ortamı sağlayın. </li></ol> Monosit türevli makrofajlar 2. Primer Kültürleri (MDM), <ol><li> 1x PBS ile buffycoat (25 mi) seyreltin. Dikkatlice oda sıcaklığında, 20 dakika boyunca 1600 x g&#39;de Ficoll / leucosep tüp ve santrifüj yüklemek. </li><li> Yeni bir tüp içine arayüzde monositleri toplayın. 10 ml ile yıkayın 3x PBS% 0.1 etılendiamıntetra içeren 1x1,000 xg, 370 x g ve 10 dakika her biri için 160 x g ve daha sonra bir kez 1 x PBS içinde tek başına 160 x g&#39;de, 10 dakika boyunca arka arkaya santrifüjleme ile asetik asit (EDTA). </li><li> Serumsuz 5 mi RPMI-1640 ortamı içinde hücre pelletini ve kap başına 1 x 10 6 hücre yoğunluğunda 35 mm&#39;lik kaplara hücreleri tohum. </li><li> Inkübatörde 90 dakika boyunca çökelme sonra yapışmayan hücreler ihtiva eden üst sıvıyı atmak. 1 ml PBS ile, monositlerin 3x oluşan yapışmış olan hücrelerin yıkayın; Daha sonra, daha önce serumsuz hücreler% 10 gliserol içeren taze ortam (h / h) insan serumunun 1 ml. </li><li> 6 günlük bir kültürden sonra, alternatif farklı makrofajlar (M2) elde rekombinant insan IL-4 (15 ng / ml) ekleyin ve 6 gün için muhafaza etmek. Daha sonra, M1, makrofajlar elde etmek üzere 4 saat 12. günde lipopolisakarit (100 ng / ml) ile farklılaştırılmış makrofajlar tedavi. </li></ol> 2D Elektroforez için M1 ve M2 Makrofaj Proteinlerin 3. Ekstraksiyon <ol><li> Yıkama macrophyaşları 25 mM Tris, 30 mM Tris pH 8, 4,% 3 ihtiva eden tampon içinde pH 7,4 ve hurda ile üç kez - [(3-kolamidopropil) -dimetilamonyo] -1-propansülfonat (CHAPS), 2M tioüre ve 7 M üre. </li><li> -20 ° C&#39;de bir buz ve deposunda 5 dakika için 1.5 ml mikrosantrifüj tüplerine için müsait bir mikser kullanılarak Hücreleri. </li><li> Ticari bir Bradford reaktifi kullanılarak protein konsantrasyonunu belirleyin. Kullanılana kadar -20 ° C&#39;de, proteinlerin 100 ul alikotları saklayın. </li></ol> 4. izoelektro-odaklanma NOT: Karanlıkta bütün etiketleme prosedürleri uygulayın. <ol><li> 37 ° C&#39;de 1 saat boyunca 2 mM Tris (2-karboksietil) fosfin (TCEP); ihtiva eden liziz tamponu ile 9 ul ayarlandı her bir örnek (M1 ve M2 makrofajlar) 5 ug azaltır. </li><li> 0.8 nM / ug protein konsantrasyonunda M1 özü ve M2 sülfidril-reaktif boya ekstreye siyanin 3 (Cy3) ekleyin. M1 siyanin 5 (Cy5) ilave et ve M2 0.8 nM / ug protein konsantrasyonunda alır ve inkübe37 ° C&#39;de 30 dakika karıştırıldı. </li><li> 7 M üre, 2 M tioüre, 4% CHAPS, 130 mM ditiotreitol (DTT) ve% 2 pharmalytes ihtiva eden numune tampon maddesinin eşit hacmi eklenerek reaksiyonu durdurun. </li><li> Mix bir yandan Cy5 etiketli M2 örnekleri ile M1 örnekleri Cy3 etiketli ve başka bir el Cy5 etiketli M1 örnekleri ile M2 örnekleri Cy3 etiketli. </li><li> Bir İmmobilize pH gradyanı Rehydrate (IPG) şerit (240 mm, pH 3-10 doğrusal gradyan) 7 M üre, bir İsoelektrik 2 M tiyoüre ve% 4 CHAPS odaklanarak içeren tamponda etiketli karışık örneklerin 450 ul (İEF) hücre sistemi herhangi bir akım uygulamadan 24 saat karıştırıldı. </li><li> 3 saat ve 3 saat sonunda 8,000 V 8000 V bir gradyan ile, 6 saat süre ile 1000 V bir gradyan daha sonra da 3 saat boyunca 300 volt (V) &#39;de odaklama yapın ve. </li></ol> 5. İkinci Boyut NOT: Karanlıkta bütün elektroforez işlemleri gerçekleştirin. <ol><li> Dengeleme tamponunda IPG şeritler içeren inkübe10 dakika süre ile 0.1 mM Tris-HCl (pH 8), 6 M üre,% 2 sodyum dodesil sülfat (w / v) (SDS) ve% 30 (h / h) gliserol ing. </li><li> % 12.5 PAGE jelleri üzerine, ikinci boyutta (SDS-poliakrilamid jel elektroforezi (PAGE)) için dengelenmiş IPG şerit aktarma ve agaroz, erime noktası düşük ile sağlanır. </li><li> Bromofenol mavisi önü jelin dibine ulaşana kadar, gece boyunca 300 V ve ardından, 70 V sabit gerilimde, bir Ettan-Daltsix sistemi kullanılarak 20 ° C &#39;de elektroforez yapınız. </li></ol> 6. Görüntü Alma ve Biyoinformatik Analizi <ol><li> Cy5 100 um bir piksel boyutu ile görüntü elde etmek için Cy3 için 532/580 nm ve 633/670 nm uyarım / emisyon dalga boylarında, bir DIGE Görüntüleyici tarayıcı ile iki düşük floresan cam plakalar arasına dökülmektedir Tarama jeller. </li><li> Daha önce 12 ayrıntılı olarak ticari yazılım ile görüntü analizi yapın. </li><li> Hesaplayın ve her görüntüde spot hacimleri normale. Bir pervane gibi bir normalize nokta hacmi atamaHer nokta, toplam değerinin ortion jel saptanmıştır. </li><li> Iki grup (M1 ve M2) arasındaki normalize nokta hacim değeri karşılaştırarak makrofajlar her tip protein nokta hacimlerindeki farklılıkları analiz. Değişim 1.5 kat (p &lt;0.05, tek yönlü ANOVA analizi) ise anlamlı nokta hacimleri arasındaki farkı düşünün. </li></ol>

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There are no declared conflict of interest.

Bu tarifnamede tarif edilen protokol makrofajlar, M1 (pro-enflamatuar) ve M2 (anti-iltihap) olarak iki alt-tip farklı uyaranların etkisini analiz etmek için bir yöntem göstermektedir. Daha önce 7 yayınlanan M1 ve M2 makrofajların birincil kültürleri monositlerin farklılaşması elde edilmiştir. 2D DIGE jel elektroforezi prosedür, Cy3 ve Cy5 floresans bir tarayıcı için uyarım / emisyon dalga iki kez jel tarama yapmak için SDS-PAGE için izoelektro-odaklanma, düş...

Makrofajlar, ayırt edici fonksiyonel fenotipleri elde edebiliyoruz heterojen ve plastik hücrelerdir. İn vivo olarak, bu hücreler mikrobiyal ürünler, sitokinler, vb. 1 mikro çevre signalssuch büyük bir çeşitlilik yanıt. İn vitro, pro-enflamatuar fenotip (M + -1) makrofaj lipopolisakarit (LPS) ve interlökin-4 (IL-4) gibi sitokinler tarafından anti-iltihabik bir fenotipe (M2) tarafından indüklenebilir. Dahası, makrofajlar belirli sinyalleri 2 üzerine, tersine M2 fenotip bir aktive M1 geçiş yapabilirsiniz. Fenotip bağlı olarak, makrofajlar farklı işlevlere sahip olacaktır. M1 makrofajlar mikroorganizmaları ve tümör hücrelerini öldürmeye 3, tümör nekroz faktörü α (TNF-α) gibi pro-enflamatuar sitokinleri üreten hücrelerdir. Buna karşılık, M2 makrofajlar anti-enflamasyon üreterek yara iyileşmesi ve fibrozis gibi bu enflamatuar yanıtı önlemekörneğin 3,4, TGF-β Y faktörler. Daha önce Boyum 6 uyarlanan bir teknik kullanarak 5 tarif edilen sağlıklı donörlerden elde edilen insan periferal kan tek-çekirdekli hücreleri Ficoll yoğunluk farklı santrifüjü yoluyla izole edilmiştir. Kültür makrofajlar primer kültür 7 6 gün sonra M1 veya M2 fenotip ayırt edilebilir. Protein ekspresyonu ya da ana bilgisayar patojenik veya mikrobik toksinler gibi çeşitli kontrollü uyaranlarla altında makrofajların iki alt tip arasında, protein değişikliklerin analizi, pro- ve anti-inflamatuar makrofaj özelliğe deşifre yararlı olacaktır. Proteomiks özellikle yukarı veya çeşitli uyaranlara altında insan kültürlü makrofajlar aşağı-regüle olan proteinlerin doğrudan izlenmesi için benzersiz araçlardır. Floresan boyalar gibi düşük duyarlılık ve görüntü analizi 8 gibi 2D jel elektroforezi sınırlamaları, bazı çözdük &lt;<html> / Sup&gt;. Sistein kalıntıları ile reaksiyona sokulması boyalar lizin kalıntıları 9 ile reaksiyona kıyasla tespit karşı artmış bir hassasiyet. Önceki bir çalışmada, klasik, gümüş-lekeli 2D elektroforez 11 kıyasla çok az örnekleri 10 analizinde DIGE doygunluk etiketleme yararını göstermiştir. Bu teknoloji hızlı bir makrofaj iki alt tip arasında veya aynı tipinin muamele edilmemiş ve edilmiş makrofajlar arası protein değişiklikleri analiz etmek yararlıdır. Bu proteomik tekniğin avantajları, 2 boyutlu jel 12 analiz protein boyut ve post-translasyonel modifikasyonları bilgilere erişim sahip olanlardır. Bu analiz edilebilir numunelerin sayısının sınırlandırılması, bu yüksek verimli bir yöntem değildir dikkate alınmalıdır. Kütle spektrometresi dayalı yüksek verimli deneyler gelişimi gözden olarak son 13 bu artırabilirsiniz. _content &quot;&gt; Burada elektroforez, izoelektro-odaklanma süreçleri yoluyla kültürlü makrofajlar protein çıkarılması 2D DIGE analizi gerçekleştirmek için nasıl sunmak ve yeterli 2D yazılım kullanışlılığı SDS-PAGE yanı sıra bilgi.

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>31870-074</td><td></td></tr><tr><td>PBX 10 X</td><td>Invitrogen</td><td>14200-083</td><td></td></tr><tr><td>L-glutamine-200 mM-100X</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>gentamycin 10 mg/ml</td><td>Invitrogen</td><td>15710-049</td><td></td></tr><tr><td>human serum</td><td>Invitrogen</td><td>34005100</td><td></td></tr><tr><td>Ficoll d = 1,077</td><td>ATGC</td><td>L6115</td><td></td></tr><tr><td>Leucosep</td><td>Dutscher</td><td>16760</td><td></td></tr><tr><td>&amp;nbsp;6-wells plate PRIMARIA&amp;nbsp;</td><td>Becton Dickinson</td><td>353846</td><td></td></tr><tr><td>IL-4</td><td>Promocell</td><td>B-61410</td><td></td></tr><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L-2654</td><td></td></tr><tr><td>Giemsa</td><td>Fluka</td><td>48900</td><td></td></tr><tr><td>EDTA MM372,2</td><td>Research Organics&amp;nbsp;</td><td>3.00E+01</td><td></td></tr><tr><td>Filter 0.22 &amp;micro;m</td><td>Millipore</td><td>SCGPTORE</td><td></td></tr><tr><td>100 ml cylinder</td><td>Corning</td><td>430182</td><td></td></tr><tr><td>TCEP</td><td>Interchim</td><td>UP242214</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Cy3+Cy5-reactive dye</td><td>GE Healthcare</td><td>25-8009-83</td><td></td></tr><tr><td>IPG strip 3-10 24cm</td><td>GE Healthcare</td><td>17-6002-44</td><td></td></tr><tr><td>Protean IEF cell</td><td>Bio-Rad</td><td>165-4000</td><td></td></tr><tr><td>Low-melting agarose</td><td>Invitrogen</td><td>15517-014</td><td></td></tr><tr><td>Ettan-Daltsix system</td><td>GE Healthcare</td><td>80-6485-08</td><td></td></tr><tr><td>Ettan DIGE Imager scanner</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Progenesis Samespot</td><td>Non linear dynamics</td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>15 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>CHAPS</td><td>Sigma-Aldrich</td><td>C5070</td><td></td></tr><tr><td>Ur&amp;eacute;e</td><td>Merk</td><td>108484-500</td><td></td></tr><tr><td>Thiour&amp;eacute;e</td><td>Sigma-Aldrich</td><td>T7875</td><td></td></tr><tr><td>DTT</td><td>Bio-Rad</td><td>1610611</td><td></td></tr><tr><td>APS</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Pharmalytes 3-10</td><td>GE Healthcare</td><td>17-0456-01</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3773</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>Acrylamide 40%</td><td>Bio-Rad</td><td>161-0148</td><td></td></tr><tr><td>2D clean Up</td><td>GE Healthcare</td><td>80-6454-51</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Dim&amp;eacute;thylformamide</td><td>Sigma-Aldrich</td><td>22705-6</td><td></td></tr><tr><td>electrode wicks</td><td>Bio-Rad</td><td>165-4071</td><td></td></tr></tbody></table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Uygun diferansiyel proteomik analizi gerçekleştirmek için, analiz edilecek örneklerin işlenmesi doğrulanması gerekir. Önce 5 yayınlanan sunulan örnekte, makrofaj hücre kültürü kalitesi, morfolojik ve moleküler özellikler için gereklidir. Makrofajlar monositlerin farklılaşması ve kültürün homojenliği faz mikroskobu ile takip edilmiştir. Şekil 1, M1 ve M2 makrofajların primer kültürlerinde bir örnek göstermiştir. Gösterildiği gibi, ...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

2D Elektroforez ile Makrofagların proteomik Profil

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

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JoVE Journal

Immunology and Infection

12.6K Görüntüleme.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Video: Proteomic Profiling of Macrophages by 2D Electrophoresis

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

İnsan hipofiz adenom doku Proteom analizi için kütle spektrometresi yöntemleri ile birleştiğinde iki boyutlu Jel Elektroforez

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Kanser Araştırmaları

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Hücre yüzey Proteinlerin CyDye Dige Fluor Minimal Boyalar kullanarak Seçici Etiketleme

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Biyoloji

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Düşük oksijen ortamı altında insan makrofaj polarizasyon proteomik analizi

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biyokimya

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Tüm hücre MALDI-TOF Kütle Spektrometre Makrofaj Aktivasyon Farklı Modu analiz için Doğru ve Hızlı Yöntem

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

İmmünoloji ve Enfeksiyon

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Konsensus Beyin kaynaklı protein, 2D-DIGE ve Mini 2DE İmmünoblotting Hem kullanma İnsan ve Kemirgen Beyin proteomun Çalışmaları Ekstraksiyon Protokolü

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Nörobilim

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Protein fonksiyonu için bir 2DE-Jel Spot: Kronik Lenfositik Lösemide HS1 öğrendim Ders

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Tıp

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Hücresel lizatları Multiprotein Kompleksleri Analizi Mavi Native Poliakrilamid Jel Elektroforez (BN-PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Yalıtım, karakterizasyonu ve obezite ile ilgili iltihap tarafından etkilenen dokular makrofajlar arıtma

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Keşif Odaklı Konak-Patojen Etkileşimleri için Etiketsiz Nicel Proteomik İş Akışı

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...