2-D Electrophoresis and Analysis

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en-us LUSQF04EH 2-D Electrophoresis and Analysis 2-D Electrophoresis and Analysis /webroot/web/html/lsr/solutions/applications/2d_electrophoresis /webroot/web/images/lsr/solutions/applications/2-D_electrophoresis/2-D_electrophoresis_and_analysis/application_detail/solutions_feature_2da1_2d_electro_anlys.jpg /webroot/web/images/lsr/solutions/applications/2-D_electrophoresis/2-D_electrophoresis_and_analysis/application_thumb/cat_2da1_2d_electro_icon.jpg <p>Proteomics is the large-scale study of protein characteristics and functions. The goal of proteomics research is to obtain an integrated view of normal and abnormal cellular/organismal processes at the level of their constituent proteins (for example, in terms of protein abundance, posttranslational modifications, protein-protein interactions, and their regulatory networks).</p> <p>Proteomics can be divided into two main subcategories: protein profiling (the discovery/identification of specific targets and markers) and functional proteomics (the definition of structure, interactions, and function). 2-D PAGE is particularly well suited to protein profiling studies. It also lends itself well to targeted (functional) proteomic research, where the expression/modification of particular proteins is followed during systematic treatment regimens or alteration in growth conditions.</p> <p>Protein profiling involves comparison of 2-D gels to understand various biological processes by determining the presence or absence, up or down regulation, and modification states of proteins. Profiling thus serves as a method of discovering putatively causal correlations between protein abundance or modification states and biological processes of interest. Functional proteomics aims to test specific predictions via a more detailed analysis of proteins' structures, roles, cellular locations, and interactions with other proteins.</p> General 2-D Electrophoresis / MS Workflow <p><img usemap="#Map" src="http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt12_img1.jpg" border="0" alt="" width="575" height="539" /> <map name="Map"> <area shape="rect" coords="170,21,368,154" href="/evportal/en/US/LSR/Solutions/LUSQH6HYP/Protein_Sample_Preparation_for_2-D_Electrophoresis" /> <area shape="rect" coords="370,61,570,214" href="/evportal/en/US/LSR/Solutions/LUSQLK2B7/First_Dimension_Separation" /> <area shape="rect" coords="368,216,573,376" href="/evportal/en/US/LSR/Solutions/LUSQMI97Q/Second_Dimension_Separation" /> <area shape="rect" coords="311,378,577,522" href="/evportal/en/US/LSR/Solutions/LUSQN83Q3/Visualization_-Staining-" /> <area shape="rect" coords="163,391,309,522" href="/evportal/en/US/LSR/Solutions/LUSQON470/Imaging_and_Analysis" /> <area shape="rect" coords="15,382,129,446" href="/evportal/destination/solutions?catID=LUSPPAKG4" /> </map></p> <p></p> <p>A typical proteomics experiment (such as protein expression profiling) can be broken down into a series of steps. First, the experiment is designed so that the key parameters of the study have been vetted, transcribed, and reviewed. Second, <a href="/evportal/destination/solutions?catID=LUSQJM7OP">extraction</a>, <a href="/evportal/destination/solutions?catID=LUSQKGDN">fractionation</a>, and <a href="/evportal/destination/solutions?catID=LUSQI5CZF">solubilization</a> of proteins from a cell line, tissue, or organism is carried out. This could also include processing for partial or complete depletion of high-abundance proteins for increased sensitivity of downstream analyses. Labeling of proteins with differential dyes may also be carried out to delineate effects of various treatments. In the third step, gel-based separation of proteins in mixtures is carried out in one dimension alone (SDS-PAGE) or two successive dimensions for more complex mixtures (<a href="/evportal/en/US/LSR/Solutions/LUSQLK2B7/First_Dimension_Separation">IEF</a> followed by <a href="/evportal/en/US/LSR/Solutions/LUSQMI97Q/Second_Dimension_Separation">SDS-PAGE</a>). This is followed by gel <a href="/evportal/en/US/LSR/Solutions/LUSQN83Q3/Visualization_-Staining-">staining</a> or <a href="/evportal/en/US/LSR/Solutions/LUSQON470/Imaging_and_Analysis">imaging analysis</a> to allow for visualization, isolation and relative quantitation of proteins. Protein spots/bands of interest are excised, digested with trypsin, and identified by mass spectrometry. Additional, functional characterization of identified proteins may be done by various means.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Protein Profiling <p>The initial goal of most proteomics projects is to identify and determine differential abundance of proteins or posttranslational modifications (PTMs) between samples. Once a list of differentially affected proteins or their modified variants has been established, the subsequent step is to perform a detailed analysis of individual proteins of interest. This may require their expression and purification, structural characterization, assessment of biochemical activity, identification of interacting partners, or production of antibodies for additional studies. Because these analyses are time consuming and costly, accurate identification of differentially expressed proteins is critical. Unlike shotgun proteomics experiments, 2-D gel electrophoresis followed by mass spectrometry provides direct visual confirmation of changes in protein/PTM abundance, thus providing early justification for downstream analytical steps. A few additional applications of profiling are discussed below.</p> <ul> <li><strong>Biomarker discovery</strong> &mdash; proteomic profiling allows for biomarker discovery based on the differences in protein expression levels between samples that have been analyzed on a broad scale; that is, normal vs. disease model study, protein expression knockdown by siRNA or other conditions, etc.</li> <li><strong>Product characterization</strong> &mdash; profiling purified proteins, antibodies or samples during drug development as part of product characterization to detect batch-to-batch differences. Compared to SDS-PAGE, which provides only molecular weight information of purified proteins, 2-D electrophoresis additionally enables confirmation of uniform charge states between multiple batches</li> <li><strong>Protein purification</strong> &mdash; this method can be used to isolate contaminants during protein purification. Profiling samples at each step in the process can assess the purity of the end product</li> <li><a href="/en-us/applications-technologies/host-cell-protein-hcp-analysis"><strong>Host cell protein analysis</strong></a> &mdash; this streamlined HCP workflow allows the evaluation of complex antibodies when screening biopharmaceuticals (biologics) according to regulatory guidelines</li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/application_detail/2da1_img2.jpg" alt="" width="545" height="494" /></p> <p class="caption"><strong>2-D gels of total protein extracted from HeLa cells at 48 h after transfection with eGFP siRNA (control A) or actin siRNA ACT9 (B)</strong>. Enlarged images of selected areas (red rectangles, <strong>C&ndash;H</strong>) show five differentially expressed protein spots (1&ndash;5). Spot 6 showed similar intensities in both gels and served as a reference.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Posttranslational Modification (PTM) Studies <p>A critical modulator of biological activity in the proteome is posttranslational modifications, or PTMs. 2-D electrophoresis can be used effectively to study PTMs. No other technique separates charge and size isomers of polypeptides as well as 2-D electrophoresis. Hundreds to thousands of polypeptides can be resolved in a single 2-D PAGE gel. These polypeptides can be quantified, probed with antibodies (via blotting), tested for posttranslational modifications (using antibodies/chemical stains specific for each PTM), or extracted for mass spectrometric analysis.</p> <p>After enrichment of various PTMs of interest, they can be profiled via 2-D gel electrophoresis. Some such applications are discussed below.</p> <ul> <li><strong>Phospho protein expression profiling</strong> &mdash; phosphorylated protein pIs will shift to a more acidic region on the gel. Phospho expression profiling is used in signal pathway studies</li> <li><strong>Acetylated protein expression profiling</strong> &mdash; acetylation of proteins at their amino termini or on lysine side-chains will cause them to shift to a more acidic region on the gel. Acetylation of proteins can affect protein function, interactions and subsequent or additional posttranslational modifications</li> <li><strong>Methylated protein expression profiling</strong> &mdash; methylation of lysine or arginine residues can have modest effects on protein molecular weight, but shifts their isoelectric points significantly towards the acidic spectrum. Analysis of protein methylation is particularly important in epigenetic studies</li> <li><strong>Glycosylated protein expression profiling</strong> &mdash; glycosylation is both a cotranslational and posttranslational modification. Attachment of relatively simple glycans (O-glycosylation) or more complex glycans (N-glycosylation) can have varying effects on protein molecular weight, pI, and protein functions</li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/application_detail/2da1_img3.jpg" alt="" width="396" height="209" /></p> <p class="caption">Phospho-cofilin antibody probing of the 2-D gel revealed that spot c6 was the non-phosphorylated cofilin, spots c1&ndash;c5 were phosphorylated cofilins. 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width="620" height="376"></iframe></div> </div> 6478 /templatedata/internet/documentation/data/LSR/Literature/6478_1401994020.xml 2-D Electrophoresis Workflow Brochure, Rev B 6478 H /webroot/web/pdf/lsr/literature/Bulletin_6478.pdf Literature PDF Brochures_and_Specifications 2-D Electrophoresis Workflow Brochure, Rev B No 2-D Electrophoresis Workflow Brochure, Rev B 6478 6478, 2D electrophoresis, 2-D, ReadyPrep, PROTEAN i12, ProteoMiner, Bio-Lyte, BioLyte, ReadyStrip, Criterion Dodeca, ChemiDoc, Flamingo, Silver Stain Plus, EXQuest, TGX Stain-Free, Oriole, IEF separation, isoelectric focusing 6493 /templatedata/internet/documentation/data/LSR/Literature/6493_1401994287.xml Bio-Rad’s Host Cell Protein (HCP) Workflow Quick Guide 6493 H /webroot/web/pdf/lsr/literature/Bulletin_6493.pdf Literature PDF Manuals_and_Quick_Guides Bio-Rad’s Host Cell Protein (HCP) Workflow Quick Guide No Bio-Rad’s Host Cell Protein (HCP) Workflow Quick Guide, Rev C 6493 6493, hcp, host cell, protein, 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1632043, 1632045, 1632044, 1632046, 1632047, 1646000, 1656001, 1703125, 1704155, 1704275, 1703127, 1705060, 1705061, 1708280, 1708370, 1709630, 5671071, 5671081, 5671091, 5671101, 5671111, 5671121 6040 Electrophoresis Guide, Interactive PDF, Rev C 6040 /webroot/web/pdf/lsr/literature/Bulletin_6040.pdf Literature PDF Manuals_and_Quick_Guides /webroot/web/images/general/icons/icon_pdf.gif No Electrophoresis Guide, Interactive PDF, Rev C 6040 Bulletin 2895, 6040, overview and principle, theory and product selection, one dimensional (1-D) protein separation, blue native polyacrylamide, SDS-PAGE, sodium dodecyl sulfate, sodium dodecyl sulphate, polymerization, precast, handcast resolving, stacking single-percentage gel, Laemmli (Tris-HCl), Bis-Tris, Tris-acetate, Tris-Tricine premade running and sample buffers, formulations/components, reagents, salts, isoelectric focusing (IEF), zymogram, gel casting chambers, gradient formers, sample preparation protocols, cell lysis, disruption, homogenization, protein solubilization, human suspension/monolayer cultured cells, mammalian tissue, plant leaves, microbial cultures, chromatography protein fractions, reducing, chaotropic agents, detergents, sample loading and quantitation spectrophotometric assays, recombinant and natural protein standards, prestained or unstained molecular weight markers or ladders, selecting power supplies, joule heating, separations under constant voltage, current, power, detection and analysis, total and specific protein staining, Coomassie Blue stain, fluorescent gel and silver stains, stainers, imagers, imaging systems and software, protein molecular weight (size) estimation, quantification, downstream applications, western blotting, gel drying, electroelution, spot excision (cutting), troubleshooting tips 2651 2-D Electrophoresis Workflow How-To Guide, Rev F 2651 /webroot/web/pdf/lsr/literature/Bulletin_2651.pdf Literature PDF Manuals_and_Quick_Guides /webroot/web/images/general/icons/icon_pdf.gif 2-D Electrophoresis Workflow How-To Guide No 2-D Electrophoresis Workflow How-To Guide, Rev F 2651 proteomics, bulletin 2651, two dimensional electrophoresis, LIT2651, sample preparation, 2d, analytical, proteomeworks, prefractionation, 2-d manual, 2-d guide, 2-d electrophoresis workflow, 2-d methods and product, 2d manual, 2d guide, 2d electrophoresis workflow, 2d methods and product 3097 Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B 3097 /webroot/web/pdf/lsr/literature/Bulletin_3097.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Imaging and Analysis: Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B 3097 1-D gels, gel, GS-800 Calibrated densitometer, Personal Molecular Imager system, pdquest, Pharos FX, VersaDoc, 5000, image, MP 4000, spot detection, ChemiDoc XRS, 2-d, Gel Doc XR, gels, protein, systems, 2d, PMI, proteins, 3097, bulletin 3097, PharosFX Plus, 2d electrophoresis, cutting, expression proteomics, EXQuest spot cutter, LIT3097, pdquest 2-d software, proteins staining, Quantity one software 2566 Normalizing Between 2-D Gels With PDQuest Software 2566 /webroot/web/pdf/lsr/literature/Bulletin_2566.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Normalizing Between 2-D Gels With PDQuest Software 2566 LIT2566, bulletin 2566, tds, proteomics, the discovery series software, proteomeworks 2629 2629 PDQuest Software Citations, Rev C /webroot/web/pdf/lsr/literature/Bulletin_2629.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No PDQuest Software Citations, Rev C Life Science 2629 LIT2629, proteomics, bulletin 2629, proteomeworks Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/1st Dimension- Isoelectric Focusing (IEF)/PROTEAN IEF Cell ->MT::92682b19-051c-464f-8a73-97cd256b225f##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/1st Dimension- Isoelectric Focusing (IEF)/ReadyStrip IPG Strips ->MT::56994cbe-ea1b-48d8-b7e9-bc17726c0d70##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Protein Stains/Fluorescent Stains ->MT::cb886820-1f6e-447f-8ef6-772fd81c29e3##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/2-D Buffers and Reagents/ReadyPrep 2-D starter kit ->MT::6a7bf285-fd23-43c4-bdd8-c4f8012d4aef## Life Science Research/Solutions/Technologies/2-D Electrophoresis ->MTS::LUSQG6LPT##Life Science Research/Solutions/Technologies/2-D Electrophoresis/Visualization -Staining- ->MTS::LUSQN83Q3##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4## Karen Moss 2-D Electrophoresis and Analysis <p>2-DE is an essential step in biomarker discovery workflow, whether your goal is protein characterization, purification and profiling, or posttranslational modification studies.</p> 12/08/11 01:40 PM 12/08/21 02:23 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Applications/2-D_Electrophoresis_and_Analysis N 0 Applications /en-us/applications-technologies/applications-technologies/2-d-electrophoresis-analysis?ID=LO8ESC15

Proteomics is the large-scale study of protein characteristics and functions. The goal of proteomics research is to obtain an integrated view of normal and abnormal cellular/organismal processes at the level of their constituent proteins (for example, in terms of protein abundance, posttranslational modifications, protein-protein interactions, and their regulatory networks).

Proteomics can be divided into two main subcategories: protein profiling (the discovery/identification of specific targets and markers) and functional proteomics (the definition of structure, interactions, and function). 2-D PAGE is particularly well suited to protein profiling studies. It also lends itself well to targeted (functional) proteomic research, where the expression/modification of particular proteins is followed during systematic treatment regimens or alteration in growth conditions.

Protein profiling involves comparison of 2-D gels to understand various biological processes by determining the presence or absence, up or down regulation, and modification states of proteins. Profiling thus serves as a method of discovering putatively causal correlations between protein abundance or modification states and biological processes of interest. Functional proteomics aims to test specific predictions via a more detailed analysis of proteins' structures, roles, cellular locations, and interactions with other proteins.

 

General 2-D Electrophoresis / MS Workflow

A typical proteomics experiment (such as protein expression profiling) can be broken down into a series of steps. First, the experiment is designed so that the key parameters of the study have been vetted, transcribed, and reviewed. Second, extraction, fractionation, and solubilization of proteins from a cell line, tissue, or organism is carried out. This could also include processing for partial or complete depletion of high-abundance proteins for increased sensitivity of downstream analyses. Labeling of proteins with differential dyes may also be carried out to delineate effects of various treatments. In the third step, gel-based separation of proteins in mixtures is carried out in one dimension alone (SDS-PAGE) or two successive dimensions for more complex mixtures (IEF followed by SDS-PAGE). This is followed by gel staining or imaging analysis to allow for visualization, isolation and relative quantitation of proteins. Protein spots/bands of interest are excised, digested with trypsin, and identified by mass spectrometry. Additional, functional characterization of identified proteins may be done by various means.

 

Protein Profiling

The initial goal of most proteomics projects is to identify and determine differential abundance of proteins or posttranslational modifications (PTMs) between samples. Once a list of differentially affected proteins or their modified variants has been established, the subsequent step is to perform a detailed analysis of individual proteins of interest. This may require their expression and purification, structural characterization, assessment of biochemical activity, identification of interacting partners, or production of antibodies for additional studies. Because these analyses are time consuming and costly, accurate identification of differentially expressed proteins is critical. Unlike shotgun proteomics experiments, 2-D gel electrophoresis followed by mass spectrometry provides direct visual confirmation of changes in protein/PTM abundance, thus providing early justification for downstream analytical steps. A few additional applications of profiling are discussed below.

  • Biomarker discovery — proteomic profiling allows for biomarker discovery based on the differences in protein expression levels between samples that have been analyzed on a broad scale; that is, normal vs. disease model study, protein expression knockdown by siRNA or other conditions, etc.
  • Product characterization — profiling purified proteins, antibodies or samples during drug development as part of product characterization to detect batch-to-batch differences. Compared to SDS-PAGE, which provides only molecular weight information of purified proteins, 2-D electrophoresis additionally enables confirmation of uniform charge states between multiple batches
  • Protein purification — this method can be used to isolate contaminants during protein purification. Profiling samples at each step in the process can assess the purity of the end product
  • Host cell protein analysis — this streamlined HCP workflow allows the evaluation of complex antibodies when screening biopharmaceuticals (biologics) according to regulatory guidelines

2-D gels of total protein extracted from HeLa cells at 48 h after transfection with eGFP siRNA (control A) or actin siRNA ACT9 (B). Enlarged images of selected areas (red rectangles, C–H) show five differentially expressed protein spots (1–5). Spot 6 showed similar intensities in both gels and served as a reference.

 

Posttranslational Modification (PTM) Studies

A critical modulator of biological activity in the proteome is posttranslational modifications, or PTMs. 2-D electrophoresis can be used effectively to study PTMs. No other technique separates charge and size isomers of polypeptides as well as 2-D electrophoresis. Hundreds to thousands of polypeptides can be resolved in a single 2-D PAGE gel. These polypeptides can be quantified, probed with antibodies (via blotting), tested for posttranslational modifications (using antibodies/chemical stains specific for each PTM), or extracted for mass spectrometric analysis.

After enrichment of various PTMs of interest, they can be profiled via 2-D gel electrophoresis. Some such applications are discussed below.

  • Phospho protein expression profiling — phosphorylated protein pIs will shift to a more acidic region on the gel. Phospho expression profiling is used in signal pathway studies
  • Acetylated protein expression profiling — acetylation of proteins at their amino termini or on lysine side-chains will cause them to shift to a more acidic region on the gel. Acetylation of proteins can affect protein function, interactions and subsequent or additional posttranslational modifications
  • Methylated protein expression profiling — methylation of lysine or arginine residues can have modest effects on protein molecular weight, but shifts their isoelectric points significantly towards the acidic spectrum. Analysis of protein methylation is particularly important in epigenetic studies
  • Glycosylated protein expression profiling — glycosylation is both a cotranslational and posttranslational modification. Attachment of relatively simple glycans (O-glycosylation) or more complex glycans (N-glycosylation) can have varying effects on protein molecular weight, pI, and protein functions

Phospho-cofilin antibody probing of the 2-D gel revealed that spot c6 was the non-phosphorylated cofilin, spots c1–c5 were phosphorylated cofilins. Note that the highly phosphorylated cofilin spot c1 was detectable only in the actin siRNA-treated HeLa cells.

 

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Literature
Number Description Download
6478 2-D Electrophoresis Workflow Brochure, Rev B Click to download
6493 Bio-Rad’s Host Cell Protein (HCP) Workflow Quick Guide Click to download
6040 Electrophoresis Guide, Interactive PDF, Rev C Click to download
2651 2-D Electrophoresis Workflow How-To Guide, Rev F Click to download
3097 Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B Click to download
2566 Normalizing Between 2-D Gels With PDQuest Software Click to download
2629 PDQuest Software Citations, Rev C Click to download
 
 
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The goal of proteomics research is to obtain an integrated view of normal and abnormal cellular/organismal processes at the level of their constituent proteins (for example, in terms of protein abundance, posttranslational modifications, protein-protein interactions, and their regulatory networks).</p> <p>Proteomics can be divided into two main subcategories: protein profiling (the discovery/identification of specific targets and markers) and functional proteomics (the definition of structure, interactions, and function). 2-D PAGE is particularly well suited to protein profiling studies. It also lends itself well to targeted (functional) proteomic research, where the expression/modification of particular proteins is followed during systematic treatment regimens or alteration in growth conditions.</p> <p>Protein profiling involves comparison of 2-D gels to understand various biological processes by determining the presence or absence, up or down regulation, and modification states of proteins. Profiling thus serves as a method of discovering putatively causal correlations between protein abundance or modification states and biological processes of interest. Functional proteomics aims to test specific predictions via a more detailed analysis of proteins' structures, roles, cellular locations, and interactions with other proteins.</p> General 2-D Electrophoresis / MS Workflow <p><img usemap="#Map" src="http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt12_img1.jpg" border="0" alt="" width="575" height="539" /> <map name="Map"> <area shape="rect" coords="170,21,368,154" href="/evportal/en/US/LSR/Solutions/LUSQH6HYP/Protein_Sample_Preparation_for_2-D_Electrophoresis" /> <area shape="rect" coords="370,61,570,214" href="/evportal/en/US/LSR/Solutions/LUSQLK2B7/First_Dimension_Separation" /> <area shape="rect" coords="368,216,573,376" href="/evportal/en/US/LSR/Solutions/LUSQMI97Q/Second_Dimension_Separation" /> <area shape="rect" coords="311,378,577,522" href="/evportal/en/US/LSR/Solutions/LUSQN83Q3/Visualization_-Staining-" /> <area shape="rect" coords="163,391,309,522" href="/evportal/en/US/LSR/Solutions/LUSQON470/Imaging_and_Analysis" /> <area shape="rect" coords="15,382,129,446" href="/evportal/destination/solutions?catID=LUSPPAKG4" /> </map></p> <p></p> <p>A typical proteomics experiment (such as protein expression profiling) can be broken down into a series of steps. First, the experiment is designed so that the key parameters of the study have been vetted, transcribed, and reviewed. Second, <a href="/evportal/destination/solutions?catID=LUSQJM7OP">extraction</a>, <a href="/evportal/destination/solutions?catID=LUSQKGDN">fractionation</a>, and <a href="/evportal/destination/solutions?catID=LUSQI5CZF">solubilization</a> of proteins from a cell line, tissue, or organism is carried out. This could also include processing for partial or complete depletion of high-abundance proteins for increased sensitivity of downstream analyses. Labeling of proteins with differential dyes may also be carried out to delineate effects of various treatments. In the third step, gel-based separation of proteins in mixtures is carried out in one dimension alone (SDS-PAGE) or two successive dimensions for more complex mixtures (<a href="/evportal/en/US/LSR/Solutions/LUSQLK2B7/First_Dimension_Separation">IEF</a> followed by <a href="/evportal/en/US/LSR/Solutions/LUSQMI97Q/Second_Dimension_Separation">SDS-PAGE</a>). This is followed by gel <a href="/evportal/en/US/LSR/Solutions/LUSQN83Q3/Visualization_-Staining-">staining</a> or <a href="/evportal/en/US/LSR/Solutions/LUSQON470/Imaging_and_Analysis">imaging analysis</a> to allow for visualization, isolation and relative quantitation of proteins. Protein spots/bands of interest are excised, digested with trypsin, and identified by mass spectrometry. Additional, functional characterization of identified proteins may be done by various means.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Protein Profiling <p>The initial goal of most proteomics projects is to identify and determine differential abundance of proteins or posttranslational modifications (PTMs) between samples. Once a list of differentially affected proteins or their modified variants has been established, the subsequent step is to perform a detailed analysis of individual proteins of interest. This may require their expression and purification, structural characterization, assessment of biochemical activity, identification of interacting partners, or production of antibodies for additional studies. Because these analyses are time consuming and costly, accurate identification of differentially expressed proteins is critical. Unlike shotgun proteomics experiments, 2-D gel electrophoresis followed by mass spectrometry provides direct visual confirmation of changes in protein/PTM abundance, thus providing early justification for downstream analytical steps. A few additional applications of profiling are discussed below.</p> <ul> <li><strong>Biomarker discovery</strong> &mdash; proteomic profiling allows for biomarker discovery based on the differences in protein expression levels between samples that have been analyzed on a broad scale; that is, normal vs. disease model study, protein expression knockdown by siRNA or other conditions, etc.</li> <li><strong>Product characterization</strong> &mdash; profiling purified proteins, antibodies or samples during drug development as part of product characterization to detect batch-to-batch differences. Compared to SDS-PAGE, which provides only molecular weight information of purified proteins, 2-D electrophoresis additionally enables confirmation of uniform charge states between multiple batches</li> <li><strong>Protein purification</strong> &mdash; this method can be used to isolate contaminants during protein purification. Profiling samples at each step in the process can assess the purity of the end product</li> <li><a href="/en-us/applications-technologies/host-cell-protein-hcp-analysis"><strong>Host cell protein analysis</strong></a> &mdash; this streamlined HCP workflow allows the evaluation of complex antibodies when screening biopharmaceuticals (biologics) according to regulatory guidelines</li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/application_detail/2da1_img2.jpg" alt="" width="545" height="494" /></p> <p class="caption"><strong>2-D gels of total protein extracted from HeLa cells at 48 h after transfection with eGFP siRNA (control A) or actin siRNA ACT9 (B)</strong>. Enlarged images of selected areas (red rectangles, <strong>C&ndash;H</strong>) show five differentially expressed protein spots (1&ndash;5). Spot 6 showed similar intensities in both gels and served as a reference.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Posttranslational Modification (PTM) Studies <p>A critical modulator of biological activity in the proteome is posttranslational modifications, or PTMs. 2-D electrophoresis can be used effectively to study PTMs. No other technique separates charge and size isomers of polypeptides as well as 2-D electrophoresis. Hundreds to thousands of polypeptides can be resolved in a single 2-D PAGE gel. These polypeptides can be quantified, probed with antibodies (via blotting), tested for posttranslational modifications (using antibodies/chemical stains specific for each PTM), or extracted for mass spectrometric analysis.</p> <p>After enrichment of various PTMs of interest, they can be profiled via 2-D gel electrophoresis. Some such applications are discussed below.</p> <ul> <li><strong>Phospho protein expression profiling</strong> &mdash; phosphorylated protein pIs will shift to a more acidic region on the gel. Phospho expression profiling is used in signal pathway studies</li> <li><strong>Acetylated protein expression profiling</strong> &mdash; acetylation of proteins at their amino termini or on lysine side-chains will cause them to shift to a more acidic region on the gel. Acetylation of proteins can affect protein function, interactions and subsequent or additional posttranslational modifications</li> <li><strong>Methylated protein expression profiling</strong> &mdash; methylation of lysine or arginine residues can have modest effects on protein molecular weight, but shifts their isoelectric points significantly towards the acidic spectrum. Analysis of protein methylation is particularly important in epigenetic studies</li> <li><strong>Glycosylated protein expression profiling</strong> &mdash; glycosylation is both a cotranslational and posttranslational modification. Attachment of relatively simple glycans (O-glycosylation) or more complex glycans (N-glycosylation) can have varying effects on protein molecular weight, pI, and protein functions</li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/application_detail/2da1_img3.jpg" alt="" width="396" height="209" /></p> <p class="caption">Phospho-cofilin antibody probing of the 2-D gel revealed that spot c6 was the non-phosphorylated cofilin, spots c1&ndash;c5 were phosphorylated cofilins. Note that the highly phosphorylated cofilin spot c1 was detectable only in the actin siRNA-treated HeLa cells.</p> <div class="top"><a href="#helptop">Back to Top</a></div> <div class="videowrap"> <div class="videoImg"><a title="2-D Video Tutorial" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/electrophoresis/product_overlay/global/2-d_tutorial_video.html' );" href="javascript:void(0);"><img src="/webroot/web/images/lsr/support/tutorials/global/2d_tutorial.jpg" alt="" /></a></div> <div class="videoDesc"><a title="2-D Video Tutorial" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/electrophoresis/product_overlay/global/2-d_tutorial_video.html' );" href="javascript:void(0);">2-D Video Tutorial</a><br /> How to run a 2-D Gel from start to finish.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="PDQuest Software Tutorial" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pdquest_software_tutorial.html' );" href="javascript:void(0);"><img src="/webroot/web/images/lsr/support/tutorials/global/ov_pdquest2.jpg" alt="" /></a></div> <div class="videoDesc"><a title="PDQuest Software Tutorial" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pdquest_software_tutorial.html' );" href="javascript:void(0);">PDQuest Software Tutorial</a><br />PDQuest new experiment workflow.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="PDQuest Software Tutorials &mdash; 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height: 51px; border: 0px none;" src="/webroot/web/images/lsr/products/electrophoresis/product_overlay_icons/global/PROTEAN_i12_IEF_System_Tour_ov_tn.jpg" alt="" /></a></div> <div class="videoDesc"><a title="PROTEAN i12 IEF System &mdash; Web Application" onclick="javascript:openElementOverlay('i12WebApp');" href="javascript:void (0);">PROTEAN i12 IEF System</a> <br />Web Application</div> <div class="clear">&nbsp;</div> </div> <div id="i12WebApp" style="display: none;"> <div class="overlay_contents" style="height: 383px;"> <div class="overlay-head">PROTEAN i12 IEF System</div> <iframe src="http://www.youtube.com/embed/uNnpnhZ1v6E?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" width="620" height="376"></iframe></div> </div> 6478 /templatedata/internet/documentation/data/LSR/Literature/6478_1401994020.xml 6493 /templatedata/internet/documentation/data/LSR/Literature/6493_1401994287.xml 6040 2651 3097 2566 2629 Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/1st Dimension- Isoelectric Focusing (IEF)/PROTEAN IEF Cell ->MT::92682b19-051c-464f-8a73-97cd256b225f##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/1st Dimension- Isoelectric Focusing (IEF)/ReadyStrip IPG Strips ->MT::56994cbe-ea1b-48d8-b7e9-bc17726c0d70##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Protein Stains/Fluorescent Stains ->MT::cb886820-1f6e-447f-8ef6-772fd81c29e3##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/2D-Electrophoresis/2-D Buffers and Reagents/ReadyPrep 2-D starter kit ->MT::6a7bf285-fd23-43c4-bdd8-c4f8012d4aef## Life Science Research/Solutions/Technologies/2-D Electrophoresis ->MTS::LUSQG6LPT##Life Science Research/Solutions/Technologies/2-D Electrophoresis/Visualization -Staining- ->MTS::LUSQN83Q3##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4## Karen Moss 2-D Electrophoresis and Analysis <p>2-DE is an essential step in biomarker discovery workflow, whether your goal is protein characterization, purification and profiling, or posttranslational modification studies.</p> 12/08/11 01:40 PM 12/08/21 02:23 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Applications/2-D_Electrophoresis_and_Analysis N 0

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