Vertical Streaking

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en-us LUSQS649 Vertical Streaking Vertical Streaking /webroot/web/html/lsr/solutions/technologies/2d_electrophoresis <p>Welcome to the 2-D Doctor&trade; section on vertical streaking. The 2-D Doctor is a self-help guide that enables you to troubleshoot your 2-D gel issues. Here you will find solutions to the problem of vertical streaking of 2-D gels.</p> <p>Other sections in the 2-D Doctor:</p> <ul> <li><a href="/evportal/destination/solutions?catID=LUSQRG30E">Horizontal Streaking</a></li> <li><a href="/evportal/destination/solutions?catID=LUSQTG84">Irregular Spot Patterns</a></li> </ul> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Problems and Solutions</h3> </div> <div class="contentroundmidwrap"> <div class="contentroundmid gels"> <p>Click on the thumbnail that is most representative of your own gel to find out the probable cause of and specific solutions to your problem.</p> <table class="pd_table" border="0"> <tbody> <tr> <td> <div><a href="#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel1.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel1">Vertical Streaking at the End of the Gel</a></p> </div> </td> <td> <div><a href="#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel2.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel2">Vertical Spot Streaking</a></p> </div> </td> <td> <div><a href="#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel3.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel3">Vertical Streaking Down Length of Gel</a></p> </div> </td> </tr> <tr> <td> <div><a href="#gel4"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel4.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel4">Isolated Vertical Blank Stripe</a></p> </div> </td> <td> <div><a href="#gel5"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel5.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel5">Vertical Streaking Across Entire Gel</a></p> </div> </td> <td> <div><a href="#gel6"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel6.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel6">Large Vertical Blank Stripe</a></p> </div> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p style="margin-top: 10px;">For additional help, you can also view <a href="#related_content">videos</a> and browse <a href="#related_content">FAQs</a>.</p> <div class="contentroundwrap"><a name="gel1"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking at the End of the Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img1.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td width="28%" valign="top"><strong>Likely Cause</strong></td> <td width="72%" valign="top">Protein aggregation or precipitation</td> </tr> <tr> <td rowspan="5" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Dilute the sample.</td> </tr> <tr> <td width="72%" valign="top">Perform a protein assay prior to IEF to ensure correct protein load. The total amount of protein that should be loaded onto an IPG strip usually depends on the length of the strip and the stain that will be used to visualize the results.</td> </tr> <tr> <td width="72%" valign="top">A distinct vertical protein front can often be observed in the middle range of 2-D gels when using cup loading. During focusing, all proteins move out of the sample cup in the same direction, causing a distinct vertical band. Increase the total volt-hours to minimize this effect and attain more complete focusing.</td> </tr> <tr> <td width="72%" valign="top">Load your sample using in-gel rehydration.</td> </tr> <tr> <td width="72%" valign="top">Prolong the time on the initial low-voltage steps and increase the voltage gradually. For example: <br /> <br /> <table class="pd_table pd_gridlines" style="margin-left: 0" border="0"> <tbody> <tr bgcolor="#666666"> <td class="small"><strong><span style="color: #ffffff;">Step</span></strong></td> <td class="small"><strong><span style="color: #ffffff;">Voltage</span></strong></td> <td class="small"><strong><span style="color: #ffffff;">Time</span></strong></td> </tr> <tr> <td class="small">1</td> <td class="small">150 V</td> <td class="small">&gt;3 hr</td> </tr> <tr> <td class="small">2</td> <td class="small">300 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">3</td> <td class="small">600 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">4</td> <td class="small">1,200 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">5</td> <td class="small">1,200&ndash;10,000 V linear gradient</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">6</td> <td class="small">10,000 V</td> <td class="small">Steady-state</td> </tr> </tbody> </table> <p>&nbsp;</p> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel2"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Spot Streaking</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img2.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td rowspan="4" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Iodoacetamide is too old</td> </tr> <tr> <td width="72%" valign="top">Equilibration time was too short</td> </tr> <tr> <td width="72%" valign="top">SDS gel has wrong pH</td> </tr> <tr> <td width="72%" valign="top">SDS concentration was too low in equilibration solution</td> </tr> <tr> <td rowspan="4" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Make sure all reagents are fresh, including Iodoacetamide and DTT.</td> </tr> <tr> <td width="72%" valign="top">Prolong the equilibration step. Carry out equilibration for as long as 40 min if necessary.</td> </tr> <tr> <td width="72%" valign="top">Ensure that the pH of the Tris buffer used for SDS-gel casting is 8.8. The wrong pH decreases the mobility of the protein-SDS complexes and causes vertical streaks of protein.</td> </tr> <tr> <td width="72%" valign="top">Ensure that the SDS concentration is at least 2% (w/v) in the equilibration solution.</td> </tr> <tr> <td rowspan="7" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&amp;_pageLabel=productsPage&amp;catID=c1b29451-ee38-43f4-bfa1-c7f36dcd662e">Bio-Rad precast gels</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2107">ReadyPrep&trade; 2-D starter kit equilibration buffer I</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2108">ReadyPrep 2-D starter kit equilibration buffer II</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0798">Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2109">Bio-Rad iodoacetamide, 30g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0610">Bio-Rad dithiothreitol, 1g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0611">Bio-Rad dithiothreitol, 5g</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel3"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking Down Length of Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img3.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td rowspan="2" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Improper IPG strip rehydration</td> </tr> <tr> <td width="72%" valign="top">Insufficient rehydration solution</td> </tr> <tr> <td rowspan="2" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Ensure rehydration volumes are correct for the IPG strip length being used. If loading protein onto the IPG strip by including the sample in the rehydration solution, do not exceed the recommended volume, as doing so may result in incomplete entry of proteins into the IPG strip.</td> </tr> <tr> <td width="72%" valign="top">If the sample appears unevenly distributed, or if areas of the strip are not wetted with sample, slide the strip back and forth several times along the length of the channel in the focusing tray.</td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel4"></a> <div class="contentroundtop"> <h3><strong>Problem: Isolated Vertical Blank Stripe</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img4.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" width="28%" valign="top"><strong>Likely Causes</strong></td> <td width="72%" valign="top">Air bubbles trapped between the IPG strip and second-dimension gel</td> </tr> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Ensure that the 2-D gel has a straight, level top edge and that the IPG strip is in direct contact with the 2-D gel along its entire length.</td> </tr> <tr> <td width="72%" valign="top">Use a 0.5% agarose overlay solution to prevent the IPG strip from coming loose or moving. To minimize the number of bubbles in the overlay, melt the agarose overlay solution completely prior to loading.</td> </tr> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2092">PROTEAN<sup>&reg;</sup> Plus overlay agarose</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2111">ReadyPrep overlay agarose</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel5"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking Across Entire Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img5.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Leaking of the upper buffer reservoir (cathode) of a vertical electrophoresis unit</td> </tr> <tr> <td width="72%" valign="top">Improper equilibration</td> </tr> <tr> <td width="72%" valign="top">Old DTT and Iodoacetamide preparations</td> </tr> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Prior to inserting the gel(s) into the vertical electrophoresis cell, wet the gaskets of the electrophoresis chamber with water or use a small amount of vacuum grease.</td> </tr> <tr> <td width="72%" valign="top">Increase the equilibration time to 15 min.</td> </tr> <tr> <td width="72%" valign="top">Use fresh reagents for the equilibration step.</td> </tr> <tr> <td class="list_title" rowspan="6" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0798">Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2107">ReadyPrep 2-D starter kit equilibration buffer I</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2108">ReadyPrep 2-D starter kit equilibration buffer II</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2109">Bio-Rad iodoacetamide, 30g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0610">Bio-Rad dithiothreitol, 1g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0611">Bio-Rad dithiothreitol, 5g</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel6"></a> <div class="contentroundtop"> <h3><strong>Problem: Large Vertical Blank Stripe</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img6.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Interfering substances; impurities in the rehydration/sample solution</td> </tr> <tr> <td width="72%" valign="top">Air bubble trapped between the IPG strip and second-dimension gel</td> </tr> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Remove contaminants such as salts.</td> </tr> <tr> <td width="72%" valign="top">Use high-quality reagents and chemicals for electrophoresis to minimize the risk of impurities. Replace chemicals of questionable or unknown shelf life, origin, or quality, as these products can also contribute to poor 2-D results.</td> </tr> <tr> <td width="72%" valign="top">Ensure no bubbles are trapped between the IPG strip and the gel surface.</td> </tr> <tr> <td class="list_title" rowspan="4" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=bbf3a0be-188f-45c7-b81e-e9706ec77c02">ReadyPrep 2-D cleanup kit</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2106">ReadyPrep 2-D starter kit rehydration/sample buffer</a></td> </tr> <tr> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=749eaa83-8fb4-4854-bc8a-84ab01195fb9">ReadyPrep protein extraction kit (total protein)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=9c6fff0c-677e-440d-9795-49d17aa08aae">ReadyPrep reduction-alkylation kit</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p><a name="related_content"></a></p> Protocols <table id="carttablealigned" class="literature_table" style="height: auto; width: 583px;" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <th>Number</th> <th>Description</th> <th class="options">Options</th> </tr> <tr> <td>6222</td> <td>IPG Equilibration for the Second Dimension, Placement and Agarose Embedding of IPG Strips<br /></td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6222.pdf" target="_blank"><span>Click to download</span></a></td> </tr> <tr> <td>6236</td> <td>Using Precison Plus Protein Standard Plugs<br /></td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6236.pdf" target="_blank"><span>Click to download</span></a></td> </tr> </tbody> </table> <div class="videowrap"> <div class="videoImg"><a title="Avoiding Horizontal Streaks" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/2d_electrophoresis_avoidinghorzstreakl/2d_streaking_final.htm', 'StatusBar', 'toolbar=no, resizable=no, width=810, height=575');" href="javascript:void(0);"> <img style="border: medium none;" src="/webroot/web/images/lsr/support/tutorials/global/2d_avoiding.png" alt="" /></a></div> <div class="videoDesc"><a title="Avoiding Horizontal Streaks" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/2d_electrophoresis_avoidinghorzstreakl/2d_streaking_final.htm', 'StatusBar', 'toolbar=no, resizable=no, width=810, height=575');" href="javascript:void(0);">2-D Electrophoresis: Avoiding Horizontal Streaks</a><br /> Horizontal streaks in 2-D gels are the result of poor protein separation during isoelectric focusing (IEF). This tutorial discusses the most common sources of horizontal streaks in 2-D gels: problems with sample preparation and inappropriate IEF conditions.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <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 style="border:none" 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> 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 2587 2587 High-Performance 2-D Gel Electrophoresis Using Narrow pH-Range ReadyStrip IPG Strips, Rev C /webroot/web/pdf/lsr/literature/Bulletin_2587.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No High-Performance 2-D Gel Electrophoresis Using Narrow pH-Range ReadyStrip IPG Strips, Rev C Life Science 2587 bulletin 2587, LIT2587, proteomeworks 2670 2670 Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen <i>Flavobacterium psychrophilum,</i> Using a 2-D Electrophoretic Approach /webroot/web/pdf/lsr/literature/Bulletin_2670.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen <i>Flavobacterium psychrophilum,</i> Using a 2-D Electrophoretic Approach Life Science 2670 UNO Sphere, bulletin 2670, LIT2670 2740 Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A 2740 /webroot/web/pdf/lsr/literature/Bulletin_2740.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A 2740 LIT2740, bulletin 2740 2778 2778 Focusing Strategy and Influence of Conductivity on Isoelectric Focusing in Immobilized pH Gradients, Rev A /webroot/web/pdf/lsr/literature/Bulletin_2778.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Focusing Strategy and Influence of Conductivity on Isoelectric Focusing in Immobilized pH Gradients, Rev A Life Science 2778 LIT2778, 2-d electrophoresis, ief 2859 2859 Combination of 2-D Gel and Liquid-Phase Electrophoretic Separations As Proteomic Tools in Neuroscience, Rev A /webroot/web/pdf/lsr/literature/Bulletin_2859.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Combination of 2-D Gel and Liquid-Phase Electrophoretic Separations As Proteomic Tools in Neuroscience, Rev A Life Science 2859 sample preparation, LIT2859, prep cell, rotofor, bulletin 2859 3098 Expression Proteomics Overview Brochure, Rev B 3098 /webroot/web/pdf/lsr/literature/Bulletin_3098.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Expression Proteomics Overview Brochure, Rev B 3098 2-D gel electrophoresis, 2D, mass spectrometry, membrane protein isolation, proteins, 2DGE, 3098, LIT3098, MALDI, ion-exchange chromatography, sample preparation, tools, 2-D electrophoresis, PDQuest image analysis software, bulletin 3098, imaging, 2 D, LC-MS/MS, preparative-scale, protein 5545 5545 Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains (Poster), Rev A /webroot/web/pdf/lsr/literature/5545RevA.pdf Literature PDF Scientific Posters /webroot/web/images/general/icons/icon_pdf.gif No Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains (Poster), Rev A Life Science 5545 LIT5545, 161-0492, 1610491, 1610492, 161-0490, 161-0491, 1610490 6097 PROTEAN i12 IEF System Brochure, Rev A 6097 H /webroot/web/pdf/lsr/literature/Bulletin_6097A.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No PROTEAN i12 IEF System Brochure, Rev A 6097 Bulletin 6097, 6138, 6139, 6140, 6142, two-dimensional gel electrophoresis, 2-D protein separation; 2DGE; isoelectric focusing (IEF); SDS-PAGE; broad, narrow and micro range ReadyStrip immobilized pH gradient (IPG) strips; sample rehydration method; guidelines for choice of IPG strips, rehydration method, IPG strip orientation, and loading method; focusing set up using gel side down, gel side up or gel side up with cup loading; creating, editing and programming run protocols; monitoring runs; saving, exporting and analyzing data and results; individual lane control; run multiple lanes with different samples, protocols and pH gradients; optimization in fewer runs; perform multiple experiments at once; 12 separately programmable power supplies; simultaneously optimize as many as 12 conditions; precise current limit delivered; touch-screen user interface; i12 Reporter; free web-based application; obtain reproducible data and extended separation over multiple pH ranges; narrow and micro pH ranges provide enhanced resolution; tight gel-length tolerances guarantee pH consistency; 164-6000, 1646000, 164-6001, 1646001, 163-2000, 1632000, 163-2001, 1632001, 163-2002, 1632002, 163-2003, 1632003, 163-2004, 1632004, 163-2005, 1632005, 163-2028, 1632028, 163-2029, 1632029, 163-2030, 1632030, 163-2031, 1632031, 163-2014, 1632014, 163-2015, 1632015, 163-2016, 1632016, 163-2017, 1632017, 163-2018, 1632018, 163-2019, 1632019, 163-2024, 1632024, 163-2025, 1632025, 163-2026, 1632026, 163-2027, 1632027, 163-2007, 1632007, 163-2008, 1632008, 163-2009, 1632009, 163-2010, 1632010, 163-2011, 1632011, 163-2012, 1632012, 163-2020, 1632020, 163-2021, 1632021, 163-2022, 1632022, 163-2023, 1632023, 163-2032, 1632032, 163-2033, 1632033, 163-2034, 1632034, 163-2035, 1632035, 163-2036, 1632036, 163-2037, 1632037, 163-2038, 1632038, 163-2039, 1632039, 163-2040, 1632040, 163-2041, 1632041, 163-2042, 1632042, 163-2043, 1632043, 163-2044, 1632044, 163-2045, 1632045, 163-2046, 1632046, 163-2047, 1632047, 163-2048, 1632048, 163-2049, 1632049, 163-2050, 1632050, 163-2051, 1632051, 163-2105, 1632105, 161-0378, 1610378 2621 PROTEAN Plus Dodeca Cell System Brochure, Rev C 2621 /webroot/web/pdf/lsr/literature/Bulletin_2621C.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No PROTEAN Plus Dodeca Cell Brochure, Rev C 2621 Bulletin 2621, second-dimension electrophoresis; 2DGE; two-dimensional (2-D) protein separation of complex mixtures of proteins; multi-casting chamber; Model 495 gradient former; AnyGel stand; Dodeca stainer; immobilized pH gradients (IPG) strips; uniform buffer temperature, electric field, high resolution via efficient cooling for large-format gels; 12-gel capacity; PROTEAN i12 IEF system; no buffer leaks; perfectly aligned plates eliminate potential current leaks; plate electrode give straight horizontal runs; easy assembly and cleanup; 165-4160, 1654160, 165-4120, 1654120, 165-4121, 1654121, 165-4122, 1654122, 165-4123, 1654123, 165-5131, 1655131, 165-4131, 1654131, 165-3400, 1653400, 165-3401, 1653401, 165-3403, 1653403, 165-3404, 1653404, 165-3405, 1653405, 165-3406, 1653406, 165-3407, 1653407, 165-3408, 1653408, 164-6000, 1646000, 164-6001, 1646001, 163-2000, 1632000, 163-2001, 1632001, 163-2002, 1632002, 163-2003, 1632003, 163-2004, 1632004, 163-2005, 1632005, 163-2028, 1632028, 163-2029, 1632029, 163-2030, 1632030, 163-2031, 1632031, 163-2014, 1632014, 163-2015, 1632015, 163-2016, 1632016, 163-2017, 1632017, 163-2018, 1632018, 163-2019, 1632019, 163-2024, 1632024, 163-2025, 1632025, 163-2026, 1632026, 163-2027, 1632027, 163-2007, 1632007, 163-2008, 1632008, 163-2009, 1632009, 163-2010, 1632010, 163-2011, 1632011, 163-2012, 1632012, 163-2020, 1632020, 163-2021, 1632021, 163-2022, 1632022, 163-2023, 1632023, 163-2032, 1632032, 163-2033, 1632033, 163-2034, 1632034, 163-2035, 1632035, 163-2036, 1632036, 163-2037, 1632037, 163-2038, 1632038, 163-2039, 1632039, 163-2040, 1632040, 163-2041, 1632041, 163-2042, 1632042, 163-2043, 1632043, 163-2044, 1632044, 163-2045, 1632045, 163-2046, 1632046, 163-2047, 1632047, 163-2048, 1632048, 163-2049, 1632049, 163-2050, 1632050, 163-2051, 1632051, 161-0378, 1610378 3095 2-D Electrophoresis: Tools for Rapid, High-Resolution Protein Separations Brochure, Rev B 3095 /webroot/web/pdf/lsr/literature/Bulletin_3095.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No 2-D Electrophoresis: Tools for Rapid, High-Resolution Protein Separations Brochure, Rev B 3095 bulletin 3095, dimension, gradient, multi casting, resolution, sds page, sulfate-, 2-D SDS-PAGE, 2D, first-dimension separation, proteins, ReadyStrip, separation, sodium dodecyl sulfate, SYPRO Ruby, Dodeca, first, IEF, MicroRotofor, PROTEAN II, expression proteomics, IPG strips, polyacrylamide gel electrophoresis, ReadyPrep, second, stainer, AnyGel stand, casting, Mini gels, Plus, precast, Ready Strip, sdspage, sulphate-, Blue R-250, LIT3095, Micro Rotofor, multi-casting, premixed buffer, Bio-Safe Coomassie, Criterion gel, Flamingo fluorescent, Mini-PROTEAN Tetra cell, multi-cell, Rotofor, silver, Model 491 Prep, Molecular Imager PharosFX, Ready Gel, second-dimension, stains, sulphate 3096 Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B 3096 /webroot/web/pdf/lsr/literature/Bulletin_3096.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure No Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B 3096 bio-spin, dynamic range reduction, sample prep, preparation, prep cell, rotofor, enrichment of low-abundance, isoelectric focusing, lit3096, membrane i, micro bio spin columns, model 491, proteins, truview cuvettes, cell lysis, expression proteomics, mini, prep, proteominer kits, quick start bradford assay, ready prep, readyprep, 2-d electrophoresis, affi-gel blue, albumin, aurum, chemical reagents, ief, microrotofor, protean ii, rc dc, smartspec plus, spectrophotometer, 165-2435, 1652435 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 268441258 268441565 268441566 268441989 268441989 268438804 268439702 1987 Life Science Research/Products/Protein Sample Preparation/Protein Sample Cleanup/ReadyPrep 2-D Cleanup Kit ->MT::bbf3a0be-188f-45c7-b81e-e9706ec77c02##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/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/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/Protein Standards/Recombinant Standards (Ladders)/Precision Plus Protein Standard Plugs ->MT::0c7564b9-7eec-48d8-b071-068a67e04ce4##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Midi Format 1D-Electrophoresis Systems/Criterion Cell Systems ->MT::b94e30f5-0f2a-452d-a3a0-825e8205b476## Life Science Research/Solutions/Applications/2-D Electrophoresis and Analysis ->MTS::LUSQF04EH## Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Detection and Analysis/Protein Staining ->MTS::LUSPMPE8Z##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4## Karen Moss 12/23/11 02:19 PM 12/23/21 02:26 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/Technologies/2-D_Electrophoresis N 0 Troubleshooting 2-D Electrophoresis Gels with 2-D Doctor&trade; /en-us/applications-technologies/applications-technologies/vertical-streaking?ID=LUSQQRB9O

Welcome to the 2-D Doctor™ section on vertical streaking. The 2-D Doctor is a self-help guide that enables you to troubleshoot your 2-D gel issues. Here you will find solutions to the problem of vertical streaking of 2-D gels.

Other sections in the 2-D Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own gel to find out the probable cause of and specific solutions to your problem.

 

For additional help, you can also view videos and browse FAQs.

Problem: Vertical Streaking at the End of the Gel

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Likely Cause Protein aggregation or precipitation
Recommended Solution(s) Dilute the sample.
Perform a protein assay prior to IEF to ensure correct protein load. The total amount of protein that should be loaded onto an IPG strip usually depends on the length of the strip and the stain that will be used to visualize the results.
A distinct vertical protein front can often be observed in the middle range of 2-D gels when using cup loading. During focusing, all proteins move out of the sample cup in the same direction, causing a distinct vertical band. Increase the total volt-hours to minimize this effect and attain more complete focusing.
Load your sample using in-gel rehydration.
Prolong the time on the initial low-voltage steps and increase the voltage gradually. For example:

Step Voltage Time
1 150 V >3 hr
2 300 V 1 hr
3 600 V 1 hr
4 1,200 V 1 hr
5 1,200–10,000 V linear gradient 1 hr
6 10,000 V Steady-state

 

 

Problem: Vertical Spot Streaking

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Likely Cause(s) Iodoacetamide is too old
Equilibration time was too short
SDS gel has wrong pH
SDS concentration was too low in equilibration solution
Recommended Solution(s) Make sure all reagents are fresh, including Iodoacetamide and DTT.
Prolong the equilibration step. Carry out equilibration for as long as 40 min if necessary.
Ensure that the pH of the Tris buffer used for SDS-gel casting is 8.8. The wrong pH decreases the mobility of the protein-SDS complexes and causes vertical streaks of protein.
Ensure that the SDS concentration is at least 2% (w/v) in the equilibration solution.
Recommended Products Bio-Rad precast gels
ReadyPrep™ 2-D starter kit equilibration buffer I
ReadyPrep 2-D starter kit equilibration buffer II
Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)
Bio-Rad iodoacetamide, 30g
Bio-Rad dithiothreitol, 1g
Bio-Rad dithiothreitol, 5g
 

Problem: Vertical Streaking Down Length of Gel

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Likely Cause(s) Improper IPG strip rehydration
Insufficient rehydration solution
Recommended Solution(s) Ensure rehydration volumes are correct for the IPG strip length being used. If loading protein onto the IPG strip by including the sample in the rehydration solution, do not exceed the recommended volume, as doing so may result in incomplete entry of proteins into the IPG strip.
If the sample appears unevenly distributed, or if areas of the strip are not wetted with sample, slide the strip back and forth several times along the length of the channel in the focusing tray.
 

Problem: Isolated Vertical Blank Stripe

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Likely Causes Air bubbles trapped between the IPG strip and second-dimension gel
Recommended Solution(s) Ensure that the 2-D gel has a straight, level top edge and that the IPG strip is in direct contact with the 2-D gel along its entire length.
Use a 0.5% agarose overlay solution to prevent the IPG strip from coming loose or moving. To minimize the number of bubbles in the overlay, melt the agarose overlay solution completely prior to loading.
Recommended Products PROTEAN® Plus overlay agarose
ReadyPrep overlay agarose
 

Problem: Vertical Streaking Across Entire Gel

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Likely Cause(s) Leaking of the upper buffer reservoir (cathode) of a vertical electrophoresis unit
Improper equilibration
Old DTT and Iodoacetamide preparations
Recommended Solution(s) Prior to inserting the gel(s) into the vertical electrophoresis cell, wet the gaskets of the electrophoresis chamber with water or use a small amount of vacuum grease.
Increase the equilibration time to 15 min.
Use fresh reagents for the equilibration step.
Recommended Products Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)
ReadyPrep 2-D starter kit equilibration buffer I
ReadyPrep 2-D starter kit equilibration buffer II
Bio-Rad iodoacetamide, 30g
Bio-Rad dithiothreitol, 1g
Bio-Rad dithiothreitol, 5g
 

Problem: Large Vertical Blank Stripe

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Likely Cause(s) Interfering substances; impurities in the rehydration/sample solution
Air bubble trapped between the IPG strip and second-dimension gel
Recommended Solution(s) Remove contaminants such as salts.
Use high-quality reagents and chemicals for electrophoresis to minimize the risk of impurities. Replace chemicals of questionable or unknown shelf life, origin, or quality, as these products can also contribute to poor 2-D results.
Ensure no bubbles are trapped between the IPG strip and the gel surface.
Recommended Products ReadyPrep 2-D cleanup kit
ReadyPrep 2-D starter kit rehydration/sample buffer
ReadyPrep protein extraction kit (total protein)
ReadyPrep reduction-alkylation kit
 

 
 
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    Literature
    Number Description Download
    2651 2-D Electrophoresis Workflow How-To Guide, Rev F Click to download
    2587 High-Performance 2-D Gel Electrophoresis Using Narrow pH-Range ReadyStrip IPG Strips, Rev C Click to download
    2670 Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen <i>Flavobacterium psychrophilum,</i> Using a 2-D Electrophoretic Approach Click to download
    2740 Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A Click to download
    2778 Focusing Strategy and Influence of Conductivity on Isoelectric Focusing in Immobilized pH Gradients, Rev A Click to download
    2859 Combination of 2-D Gel and Liquid-Phase Electrophoretic Separations As Proteomic Tools in Neuroscience, Rev A Click to download
    3098 Expression Proteomics Overview Brochure, Rev B Click to download
    5545 Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains (Poster), Rev A Click to download
    6097 PROTEAN i12 IEF System Brochure, Rev A Click to download
    2621 PROTEAN Plus Dodeca Cell System Brochure, Rev C Click to download
    3095 2-D Electrophoresis: Tools for Rapid, High-Resolution Protein Separations Brochure, Rev B Click to download
    3096 Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B Click to download
    3097 Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B Click to download
    Number Description Options
    6222 IPG Equilibration for the Second Dimension, Placement and Agarose Embedding of IPG Strips
    Click to download
    6236 Using Precison Plus Protein Standard Plugs
    Click to download
     
     
    LUSQS649 [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.224.11.137, 10.232.3.215] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/vertical-streaking] [x-amzn-trace-id] = [Root=1-5b7577f9-466f76f0c3a827fa3767f770] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSQS649] [host] = [10.232.0.21:1776] [x-request-uri] = [/en-us/applications-technologies/vertical-streaking] [connection] = [Keep-Alive] [accept-encoding] = [gzip] [user-agent] = [CCBot/2.0 (https://commoncrawl.org/faq/)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/vertical-streaking LSR LUSQS649 Vertical Streaking Vertical Streaking /webroot/web/html/lsr/solutions/technologies/2d_electrophoresis <p>Welcome to the 2-D Doctor&trade; section on vertical streaking. The 2-D Doctor is a self-help guide that enables you to troubleshoot your 2-D gel issues. Here you will find solutions to the problem of vertical streaking of 2-D gels.</p> <p>Other sections in the 2-D Doctor:</p> <ul> <li><a href="/evportal/destination/solutions?catID=LUSQRG30E">Horizontal Streaking</a></li> <li><a href="/evportal/destination/solutions?catID=LUSQTG84">Irregular Spot Patterns</a></li> </ul> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Problems and Solutions</h3> </div> <div class="contentroundmidwrap"> <div class="contentroundmid gels"> <p>Click on the thumbnail that is most representative of your own gel to find out the probable cause of and specific solutions to your problem.</p> <table class="pd_table" border="0"> <tbody> <tr> <td> <div><a href="#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel1.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel1">Vertical Streaking at the End of the Gel</a></p> </div> </td> <td> <div><a href="#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel2.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel2">Vertical Spot Streaking</a></p> </div> </td> <td> <div><a href="#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel3.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel3">Vertical Streaking Down Length of Gel</a></p> </div> </td> </tr> <tr> <td> <div><a href="#gel4"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel4.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel4">Isolated Vertical Blank Stripe</a></p> </div> </td> <td> <div><a href="#gel5"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel5.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel5">Vertical Streaking Across Entire Gel</a></p> </div> </td> <td> <div><a href="#gel6"><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img_gel6.jpg" border="0" alt="" width="120" height="120" /></a> <p class="caption"><a href="#gel6">Large Vertical Blank Stripe</a></p> </div> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p style="margin-top: 10px;">For additional help, you can also view <a href="#related_content">videos</a> and browse <a href="#related_content">FAQs</a>.</p> <div class="contentroundwrap"><a name="gel1"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking at the End of the Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img1.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td width="28%" valign="top"><strong>Likely Cause</strong></td> <td width="72%" valign="top">Protein aggregation or precipitation</td> </tr> <tr> <td rowspan="5" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Dilute the sample.</td> </tr> <tr> <td width="72%" valign="top">Perform a protein assay prior to IEF to ensure correct protein load. The total amount of protein that should be loaded onto an IPG strip usually depends on the length of the strip and the stain that will be used to visualize the results.</td> </tr> <tr> <td width="72%" valign="top">A distinct vertical protein front can often be observed in the middle range of 2-D gels when using cup loading. During focusing, all proteins move out of the sample cup in the same direction, causing a distinct vertical band. Increase the total volt-hours to minimize this effect and attain more complete focusing.</td> </tr> <tr> <td width="72%" valign="top">Load your sample using in-gel rehydration.</td> </tr> <tr> <td width="72%" valign="top">Prolong the time on the initial low-voltage steps and increase the voltage gradually. For example: <br /> <br /> <table class="pd_table pd_gridlines" style="margin-left: 0" border="0"> <tbody> <tr bgcolor="#666666"> <td class="small"><strong><span style="color: #ffffff;">Step</span></strong></td> <td class="small"><strong><span style="color: #ffffff;">Voltage</span></strong></td> <td class="small"><strong><span style="color: #ffffff;">Time</span></strong></td> </tr> <tr> <td class="small">1</td> <td class="small">150 V</td> <td class="small">&gt;3 hr</td> </tr> <tr> <td class="small">2</td> <td class="small">300 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">3</td> <td class="small">600 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">4</td> <td class="small">1,200 V</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">5</td> <td class="small">1,200&ndash;10,000 V linear gradient</td> <td class="small">1 hr</td> </tr> <tr> <td class="small">6</td> <td class="small">10,000 V</td> <td class="small">Steady-state</td> </tr> </tbody> </table> <p>&nbsp;</p> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel2"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Spot Streaking</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img2.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td rowspan="4" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Iodoacetamide is too old</td> </tr> <tr> <td width="72%" valign="top">Equilibration time was too short</td> </tr> <tr> <td width="72%" valign="top">SDS gel has wrong pH</td> </tr> <tr> <td width="72%" valign="top">SDS concentration was too low in equilibration solution</td> </tr> <tr> <td rowspan="4" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Make sure all reagents are fresh, including Iodoacetamide and DTT.</td> </tr> <tr> <td width="72%" valign="top">Prolong the equilibration step. Carry out equilibration for as long as 40 min if necessary.</td> </tr> <tr> <td width="72%" valign="top">Ensure that the pH of the Tris buffer used for SDS-gel casting is 8.8. The wrong pH decreases the mobility of the protein-SDS complexes and causes vertical streaks of protein.</td> </tr> <tr> <td width="72%" valign="top">Ensure that the SDS concentration is at least 2% (w/v) in the equilibration solution.</td> </tr> <tr> <td rowspan="7" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&amp;_pageLabel=productsPage&amp;catID=c1b29451-ee38-43f4-bfa1-c7f36dcd662e">Bio-Rad precast gels</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2107">ReadyPrep&trade; 2-D starter kit equilibration buffer I</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2108">ReadyPrep 2-D starter kit equilibration buffer II</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0798">Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2109">Bio-Rad iodoacetamide, 30g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0610">Bio-Rad dithiothreitol, 1g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0611">Bio-Rad dithiothreitol, 5g</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel3"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking Down Length of Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img3.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td rowspan="2" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Improper IPG strip rehydration</td> </tr> <tr> <td width="72%" valign="top">Insufficient rehydration solution</td> </tr> <tr> <td rowspan="2" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Ensure rehydration volumes are correct for the IPG strip length being used. If loading protein onto the IPG strip by including the sample in the rehydration solution, do not exceed the recommended volume, as doing so may result in incomplete entry of proteins into the IPG strip.</td> </tr> <tr> <td width="72%" valign="top">If the sample appears unevenly distributed, or if areas of the strip are not wetted with sample, slide the strip back and forth several times along the length of the channel in the focusing tray.</td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel4"></a> <div class="contentroundtop"> <h3><strong>Problem: Isolated Vertical Blank Stripe</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img4.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" width="28%" valign="top"><strong>Likely Causes</strong></td> <td width="72%" valign="top">Air bubbles trapped between the IPG strip and second-dimension gel</td> </tr> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Ensure that the 2-D gel has a straight, level top edge and that the IPG strip is in direct contact with the 2-D gel along its entire length.</td> </tr> <tr> <td width="72%" valign="top">Use a 0.5% agarose overlay solution to prevent the IPG strip from coming loose or moving. To minimize the number of bubbles in the overlay, melt the agarose overlay solution completely prior to loading.</td> </tr> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2092">PROTEAN<sup>&reg;</sup> Plus overlay agarose</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2111">ReadyPrep overlay agarose</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel5"></a> <div class="contentroundtop"> <h3><strong>Problem: Vertical Streaking Across Entire Gel</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img5.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Leaking of the upper buffer reservoir (cathode) of a vertical electrophoresis unit</td> </tr> <tr> <td width="72%" valign="top">Improper equilibration</td> </tr> <tr> <td width="72%" valign="top">Old DTT and Iodoacetamide preparations</td> </tr> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Prior to inserting the gel(s) into the vertical electrophoresis cell, wet the gaskets of the electrophoresis chamber with water or use a small amount of vacuum grease.</td> </tr> <tr> <td width="72%" valign="top">Increase the equilibration time to 15 min.</td> </tr> <tr> <td width="72%" valign="top">Use fresh reagents for the equilibration step.</td> </tr> <tr> <td class="list_title" rowspan="6" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0798">Resolving gel buffer (1.5 M Tris-HCl, pH 8.8)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2107">ReadyPrep 2-D starter kit equilibration buffer I</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2108">ReadyPrep 2-D starter kit equilibration buffer II</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2109">Bio-Rad iodoacetamide, 30g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0610">Bio-Rad dithiothreitol, 1g</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=161-0611">Bio-Rad dithiothreitol, 5g</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="gel6"></a> <div class="contentroundtop"> <h3><strong>Problem: Large Vertical Blank Stripe</strong></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><img src="/webroot/web/images/lsr/solutions/technologies/2-D_electrophoresis/2-D_electrophoresis/technology_detail/2dt172_img6.jpg" alt="" width="296" height="296" /></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td class="list_title" rowspan="2" width="28%" valign="top"><strong>Likely Cause(s)</strong></td> <td width="72%" valign="top">Interfering substances; impurities in the rehydration/sample solution</td> </tr> <tr> <td width="72%" valign="top">Air bubble trapped between the IPG strip and second-dimension gel</td> </tr> <tr> <td class="list_title" rowspan="3" width="28%" valign="top"><strong>Recommended Solution(s)</strong></td> <td width="72%" valign="top">Remove contaminants such as salts.</td> </tr> <tr> <td width="72%" valign="top">Use high-quality reagents and chemicals for electrophoresis to minimize the risk of impurities. Replace chemicals of questionable or unknown shelf life, origin, or quality, as these products can also contribute to poor 2-D results.</td> </tr> <tr> <td width="72%" valign="top">Ensure no bubbles are trapped between the IPG strip and the gel surface.</td> </tr> <tr> <td class="list_title" rowspan="4" width="28%" valign="top"><strong>Recommended Products</strong></td> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=bbf3a0be-188f-45c7-b81e-e9706ec77c02">ReadyPrep 2-D cleanup kit</a></td> </tr> <tr> <td width="72%" valign="top"><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&amp;vertical=LSR&amp;country=US&amp;lang=en&amp;productID=163-2106">ReadyPrep 2-D starter kit rehydration/sample buffer</a></td> </tr> <tr> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=749eaa83-8fb4-4854-bc8a-84ab01195fb9">ReadyPrep protein extraction kit (total protein)</a></td> </tr> <tr> <td width="72%" valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=9c6fff0c-677e-440d-9795-49d17aa08aae">ReadyPrep reduction-alkylation kit</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p><a name="related_content"></a></p> Protocols <table id="carttablealigned" class="literature_table" style="height: auto; width: 583px;" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <th>Number</th> <th>Description</th> <th class="options">Options</th> </tr> <tr> <td>6222</td> <td>IPG Equilibration for the Second Dimension, Placement and Agarose Embedding of IPG Strips<br /></td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6222.pdf" target="_blank"><span>Click to download</span></a></td> </tr> <tr> <td>6236</td> <td>Using Precison Plus Protein Standard Plugs<br /></td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6236.pdf" target="_blank"><span>Click to download</span></a></td> </tr> </tbody> </table> <div class="videowrap"> <div class="videoImg"><a title="Avoiding Horizontal Streaks" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/2d_electrophoresis_avoidinghorzstreakl/2d_streaking_final.htm', 'StatusBar', 'toolbar=no, resizable=no, width=810, height=575');" href="javascript:void(0);"> <img style="border: medium none;" src="/webroot/web/images/lsr/support/tutorials/global/2d_avoiding.png" alt="" /></a></div> <div class="videoDesc"><a title="Avoiding Horizontal Streaks" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/2d_electrophoresis_avoidinghorzstreakl/2d_streaking_final.htm', 'StatusBar', 'toolbar=no, resizable=no, width=810, height=575');" href="javascript:void(0);">2-D Electrophoresis: Avoiding Horizontal Streaks</a><br /> Horizontal streaks in 2-D gels are the result of poor protein separation during isoelectric focusing (IEF). This tutorial discusses the most common sources of horizontal streaks in 2-D gels: problems with sample preparation and inappropriate IEF conditions.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <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 style="border:none" 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> 2651 2587 2670 2740 2778 2859 3098 5545 6097 2621 3095 3096 3097 268441258 268441565 268441566 268441989 268441989 268438804 268439702 1987 Life Science Research/Products/Protein Sample Preparation/Protein Sample Cleanup/ReadyPrep 2-D Cleanup Kit ->MT::bbf3a0be-188f-45c7-b81e-e9706ec77c02##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/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/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/Protein Standards/Recombinant Standards (Ladders)/Precision Plus Protein Standard Plugs ->MT::0c7564b9-7eec-48d8-b071-068a67e04ce4##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Midi Format 1D-Electrophoresis Systems/Criterion Cell Systems ->MT::b94e30f5-0f2a-452d-a3a0-825e8205b476## Life Science Research/Solutions/Applications/2-D Electrophoresis and Analysis ->MTS::LUSQF04EH## Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Detection and Analysis/Protein Staining ->MTS::LUSPMPE8Z##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4## Karen Moss 12/23/11 02:19 PM 12/23/21 02:26 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/Technologies/2-D_Electrophoresis N 0
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