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Western Blot Troubleshooting | Thermo Fisher Scientific - SG
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468bd07150dbbf Source: /tf/js/dist/digitaldata.js */ (function () { "use strict"; /** * Invoking a Function as a Method * define function as object methods * @type {Object} */ window.digitalData = { user: {}, page: {}, merchandising: {}, getQueryParam : function (param) { var index, key, value, hash; var hashes = window.location.href.slice(window.location.href.indexOf('?') + 1).split('&'); for (index = 0; index < hashes.length; index++) { hash = hashes[index].split('='); key = hash[0]; value = hash[1]; if (key.toUpperCase() === param.toUpperCase()) return value; } return ''; }, getValOnce: function (value, cookieName, expiry, timeUnit) { var currentDate = new Date; var currentCookieValue; // timeUnit of 'm' is minutes represented in milliseconds // thus 86400000 = 24 hours = 1 day var interval = timeUnit == 'm' ? 60000 : 86400000; cookieName = cookieName ? cookieName : 'digitaldata_gvo'; value = value ? value : ''; expiry = expiry ? expiry : 0; currentCookieValue = this.getCookie(cookieName); if (value) { currentDate.setTime(currentDate.getTime() + expiry * interval); this.setCookie(cookieName, value, expiry == 0 ? 0 : currentDate); } return value == currentCookieValue ? '' : value; }, getCookie: function (key) { var result = ""; var cookies = document.cookie ? document.cookie.split('; ') : []; for (var i = 0; i < cookies.length; i++) { var parts = cookies[i].split('='); var name = decodeURIComponent(parts.shift()); var cookie = parts ? parts.join('=') : []; // Prevent storing or parsing of a cookie that we couldn't decode. if (cookie && key === name) { // removes surrounding quotes if (cookie.indexOf('"') === 0) { // This is a quoted cookie as according to RFC2068, unescape... cookie = cookie.slice(1, -1).replace(/\\"/g, '"').replace(/\\\\/g, '\\'); } // parse utf8 characters try { cookie = decodeURIComponent(cookie); } catch (e) {} result = cookie; // we found it, don't bother looking at other cookies break; } } return result; }, setCookie : function(name, value, numDays){ if ( !numDays ) numDays = 31; var expires = new Date(); expires.setTime(expires.getTime() + (1000 * 60 * 60 * 24 * numDays)); document.cookie = name+ '=' + escape(value) + '; path=/; expires=' + expires.toGMTString() + '; domain=.' + this.getMasterDomain() + ';'; }, /** * get the master domain based on business rules */ getMasterDomain : function () { // default the master domain var masterDomain = 'thermofisher.com'; // check for alternate domain portion of URL var regexMatch = document.location.hostname.match(/\.(thermofisher)\.(cn)/i); if (regexMatch !== null) { masterDomain = regexMatch[1] + '.' + regexMatch[2]; } return masterDomain; }, /** * Check if init has been initiated once * @type {Boolean} */ initted: false, /** * @return {Array} method returns an array of a given object's key properties */ init: function () { this.page.country = this.getCookie("CK_ISO_CODE") || 'us'; this.page.language = this.getCookie('CK_LANG_CODE') || 'en'; this.page.siteName = 'thermo'; //hard coded this.initMerchandisingData(); this.initUserData(); this.displayDigitalData(); this.formSubmitEvent(); }, //specific function to set type of the page // need specific function to keep page type separate from other page attributes setPageType: function (type) { this.setPageAttribute('type', type); }, // generic function to set page attributes setPageAttribute: function (key, value) { this.page[key] = value; }, // display digitalData object displayDigitalData: function () { }, // specific function to add an object to digitalData addDigitDataObject: function (key, object) { this[key] = object; }, addChildObjectAttribute: function(child, key, value){ this[child][key] = value; }, initUserData: function(){ var gigyaCookie = this.getCookie("identity_uid"); this.user.gigyaId = ""; this.user.loginStatus = "anonymous"; if( gigyaCookie != ""){ this.user.gigyaId = gigyaCookie; this.user.loginStatus = "logged in"; } }, /** * Check if init has been initiated once * @type {Boolean} */ merchandisingDataInitted: false, initMerchandisingData: function () { // read cid and icid value from session cookie this.merchandising.icid = this.getCookie('s_ev21_tc'); this.merchandising.cid = this.getCookie('s_cmp_tc'); // EMID value this.merchandising.emid = this.getQueryParam("emid"); // referrer by domain if (typeof document.referrer !== 'undefined' && document.referrer.length > 0) { var masterDomain = this.getMasterDomain(); var refReg = document.referrer; var referrerDomain = refReg.match(/:\/\/([^/]+)\//); if (referrerDomain !== null && referrerDomain[1].indexOf(masterDomain) == -1) { this.merchandising.referrer = referrerDomain; } } document.addEventListener('registerMerchandisingData', function (event) { for (var key in event.detail) { console.log("Listener data copied", key, event.detail[key] ); digitalData.merchandising[key] = event.detail[key]; } // Map some analytics attributes to merchandising session attributes digitalData.merchandising.sessionId = digitalData.merchandising.analyticsVisitorID; digitalData.merchandising.s_vi_low = digitalData.merchandising.analyticsVisitorIDLow; digitalData.merchandising.s_vi_high = digitalData.merchandising.analyticsVisitorIDHigh; console.log("digitalData: Captured registerMerchandisingData event"); digitalData.merchandisingDataInitted = true; // Call emailCampaignRecord if the digitalDataAnalyticsListener object is loaded if (typeof digitalDataAnalyticsListener != "undefined") { console.log("digitalData: Calling email campaign record"); digitalDataAnalyticsListener.emailCampaignRecord(); } }); }, formSubmitEvent: function () { var formId = this.getQueryParam("form-submit-id"); if ((formId) && formId !== "" && formId !== null) { var eventDetail = { detail: { form: { id: formId } } }; var formSubmitEvent = new CustomEvent('formSubmit', eventDetail); console.log('form submit event started'); document.dispatchEvent(formSubmitEvent); console.log('form submit event fired', eventDetail); //flush grepToken from Session Storage sessionStorage.removeItem('grepTokenSO'); } }, modalVideoEvent: function (videoId) { if (((videoId) && videoId !== "" && videoId !== null)) { var eventDetail = { detail: { video: { playerId: videoId } } }; var modalVideoEvent = new CustomEvent('videoPlayerLoaded', eventDetail); document.dispatchEvent(modalVideoEvent); } }, customClickEvent: function (clickDetail) { if (((clickDetail) && clickDetail !== "" && clickDetail !== null)) { var eventDetail = { detail: { CustomLink: { linkName: clickDetail } } } var customClickEvent = new CustomEvent('customLinkClicks', eventDetail); document.dispatchEvent(customClickEvent); } } } })(); window.digitalData.init(); </script> <!-- performance-begin --> <script type="text/javascript"> window.performance && window.performance.mark && window.performance.mark("header_25"); </script> <script type="text/javascript"> var localizedStrings = { "ACCEPTANCE": "Acceptance of Terms of Use", "BTN_AGREE": "Agree", "BTN_DISAGREE": "Disagree", "PROCESSING_MESSAGE" :"processing ... please wait ...", "ADD_TO_CART_QUANTITY_ERROR":"You must provide a quantity to add an item to the cart", "ADD_TO_CART_VALID_QUANTITY_ERROR":"You must provide a valid quantity to add an item to the cart", "VIEW_CART" :"View cart & checkout", "VIEW_CONTACT_DETAIL" :"View Contact Details", "ADD_TO_CART_NOTIFICATION":"Add to Cart Notification", "UPDATE_CART" :"Update Cart", "PRINCIPAL_INVESTIGATOR":"Principal Investigator", "PRINCIPAL_INVESTIGATOR_TEAM_LEADER":"Principal Investigator or Team Leader", "PRINCIPAL_INVESTIGATOR_CHIEF":"Principal Investigator or Chief", "EMAIL_ERROR_1" :"Please Provide Collegues Email", "EMAIL_ERROR_2" :"Please enter a valid colleague email address", "EMAIL_ERROR_3" :"Please Provide Your Email", "EMAIL_ERROR_4" :"Please enter a valid email address", "ERROR_OCCURED_MESSAGE" :"The following errors occured", "NEXT" :"Next", "PREVIOUS" :"Prev", "IMAGE" :"Image", "SAVE_TO_LIST_ERROR_1" :"Please enter a list name.", "SAVE_TO_LIST_ERROR_2" :"Please enter a different list name that does not include the following: ?,/,\\,%,^,!", "MINICART_ERROR_1" :"Unable to connect to eCommerce system. Please try again later.", "MINICART_ERROR_2" :"Please enter a quantity in the range of 1 to 999.", "MINICART_ERROR_3" :"Could not add item(s) to cart.", "MINICART_ERROR_4" :"Could not remove item from cart.", "MINICART_ERROR_5" :"Could not create/restore cart.", "MINICART_ERROR_6" :"Unable to retrieve cart details. Please try again later.", "MINICART_MESSAGE_1" :"Adding item(s) to cart...", "MINICART_MESSAGE_2" :"Removing item(s) from cart...", "MINICART_CART_NAME_LABEL_1" :"Recently added to the", "MINICART_NOTE_1" : "Your total price and product details can be viewed in the cart.", MINICART_SUMMARY : "Cart summary", MINICART_PRICE : "Price", MINICART_PRODUCT : "Product", LIST_PRICE : "Price", YOUR_PRICE : "Your price", WEB_PRICE: "Online offer", PRICE_ENDS: "ends", SIGN_IN: "Sign in", //New mini cart CATALOG_NUMBER: "Catalog number:", CONTACT_US: "Contact us", RFQ: "Request quote", LEARN_WHERE_TO_BUY: "Learn where to buy", CURRENCY: "USD", //End new mini cart "CHANGE_CART" :"Change cart", "VIEW_ALL" :"View All", "ITEM" :"item", "ITEMS" :"items", "CLOSE" :"Close", "SUBTOTAL" :"Subtotal", "TOTAL" :"Total", "MANAGE_CART" :"Manage Cart", "FIELD" :"field", "GLOBAL_ERROR_1" :"Please choose one or both of the options for", "GLOBAL_ERROR_2" :"is not a valid e-mail address.", "GLOBAL_ERROR_3" :"Unsupported character(s) were found in field", "GLOBAL_ERROR_4" :"and will be replaced with plain text equivalents (where available). 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alt='Troubleshooting' title='Troubleshooting'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_4e24 img{ width:100%; max-width: 710px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_4e24 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_4e24 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div><div class="text parbase section"><p>In this western blot troubleshooting section, we will help you visually identify specific and common problems on your western blots, such as high background, weak or no signal, multiple bands, uneven staining and suggest what may be causing them and some solutions to remedy them.</p><p>Request a free <a href="/sg/en/home/global/forms/life-science/western-blotting-troubleshooting-guide.html">Western blot tips, tricks and troubleshooting guide</a>.</p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt3"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt3-c0"><div container-id="cq-colctrl-lt3" comp-id="A1"><div class="well-cmp section"><div class="well well-gray-lighter well-default"><div class="well-container" style="height:320px"><div class="well-content"><div class="parsys"><div class="text parbase section"><h3>Protein gel electrophoresis</h3><ul><li><a href="#pge1">Protein bands lose resolution, lanes have streaks and are not straight</a></li><li><a href="#pge2">Viscous samples, streaks at sample lane edges, dumbbell- shaped bands, lane widening</a></li><li><a href="#pge3">Protein aggregation resulting in narrow lanes that cannot be interpreted</a></li><li><a href="#pge4">Uneven sample lanes, lane widening</a></li><li><a href="#pge5">Shadow at lane edges</a></li></ul></div></div></div></div></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt3-c1"><div container-id="cq-colctrl-lt3" comp-id="B1"><div class="well-cmp section"><div class="well well-gray-lighter well-default"><div class="well-container" style="height:320px"><div class="well-content"><div class="parsys"><div class="text parbase section"><h3>General western blotting</h3><ul><li><a href="#gwb1">Nonspecific or diffuse bands</a></li><li><a href="#gwb2">High background</a></li><li><a href="#gwb3">Weak signal or no signal</a></li></ul></div></div></div></div></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt3-c2"><div container-id="cq-colctrl-lt3" comp-id="C1"><div class="well-cmp section"><div class="well well-gray-lighter well-default"><div class="well-container" style="height:320px"><div class="well-content"><div class="parsys"><div class="text parbase section"><h3>Fluorescent western blotting</h3><ul><li><a href="#fwb1">Nonspecific or diffuse bands</a></li><li><a href="#fwb2">Weak or no signal</a></li><li><a href="#fwb3">Background issues (high, uneven, or speckled)</a></li></ul></div></div></div></div></div></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="heading section"><h2> Protein gel electrophoresis troubleshooting </h2></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b1bc"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b1bc.img.full.high.jpg/1726159643998.jpg' media="(min-width: 1px)"><img 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alt='Electrophoresis troubleshooting- bands lose resolution, are not straight' title='tb1'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b1bc img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="pge1"> Protein bands lose resolution, lanes have streaks and are not straight </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table--1483301067"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td>Too much protein loaded per lane</td><td>Reduce the sample loads. The maximum recommended sample load for optimal resolution in mini gels with 10, 12, 15, or 17 wells is 0.5 μg per band or about 10–15 μg of cell lysate per lane.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b338"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.full.high.jpg/1726159644038.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.full.high.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.320.high.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.480.high.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.320.medium.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.620.high.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.320.low.jpg/1726159644038.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_b338/foregroundimg.img.480.medium.jpg/1726159644038.jpg" alt='Protein electrophoresis troubleshooting, streaks at lane edges, dumbbell shaped bands' title='tb2'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b338 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b338 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_b338 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="pge2"> Viscous samples, streaks at sample lane edges, dumbbell-shaped bands, lane widening </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table--275922564"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="3">Excess salt (ammonium sulfate) in sample during gel electrophoresis</td><td>Perform dialysis to decrease salt concentration. Use a small dialysis device such as the <a href="/order/catalog/product/88401">Thermo Scientific Slide-A-Lyzer MINI Dialysis Device, 0.5 mL</a>.</td></tr><tr><td>Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as <a href="/order/catalog/product/88513">Thermo Scientific Pierce Protein Concentrators PES, 0.5 mL</a>.</td></tr><tr><td>Make sure that the salt concentration does not exceed 100 mM.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_c216"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.full.high.jpg/1726159644091.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.full.high.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.320.high.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.480.high.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.320.medium.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.620.high.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.320.low.jpg/1726159644091.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_c216.img.480.medium.jpg/1726159644091.jpg" alt='Protein electrophoresis troubleshooting, narrowing lanes, undefined bands' title='tb3'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_c216 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="pge3"> Protein aggregation resulting in narrow lanes that cannot be interpreted </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-979278182"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td>DNA contamination— genomic DNA in the cell lysate may cause the sample to become viscous, resulting in protein aggregation, which can affect protein migration patterns and resolution</td><td>Shear genomic DNA to reduce viscosity before loading the sample.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_ddd8"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.full.high.jpg/1726159644130.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.full.high.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.320.high.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.480.high.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.320.medium.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.620.high.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.320.low.jpg/1726159644130.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_ddd8/foregroundimg.img.480.medium.jpg/1726159644130.jpg" alt='Protein electrophoresis troubleshooting, lane widening' title='tb4'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_ddd8 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_ddd8 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_ddd8 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="pge4"> Uneven sample lanes, lane widening </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table striped" id="table--1214135166"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="3">Excess salt (sodium chloride) in sample during gel electrophoresis. High salt concentrations result in increased conductivity, which affects protein migration and can result in protein bands spreading into adjacent lanes containing samples with normal salt concentrations</td><td>Perform dialysis to decrease salt concentration. Use a small dialysis device such as the <a href="/order/catalog/product/88401">Slide-A-Lyzer MINI Dialysis Device, 0.5 mL</a>.</td></tr><tr><td>Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as <a href="/order/catalog/product/88513">Pierce Protein Concentrators PES, 0.5 mL</a>.</td></tr><tr><td>Make sure that the salt concentration does not exceed 100 mM.</td></tr><tr><td rowspan="2">High detergent concentration (e.g., SDS or Triton X-100 detergent) in gel electrophoresis.<br> Detergents form mixed micelles with the anionic detergent SDS in the gel and migrate down into the gel; they interfere with the SDS–protein binding equilibrium</td><td>Most of the nonionic detergents (e.g., Triton X-100, NP-40, and Tween 20 detergents) interfere with SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Keep the ratio of SDS to nonionic detergent at 10:1 or greater to minimize these effects.</td></tr><tr><td>Use detergent removal columns or the <a href="/order/catalog/product/89888">Thermo Scientific Pierce SDS-PAGE Sample Prep Kit</a> to remove excess detergent.</td></tr><tr><td>High concentration of RIPA (radioimmunoprecipitation assay) buffer results in widening of lanes and significant streaking during electrophoresis</td><td>Dilute samples before electrophoresis to lower the final concentration of lysis buffer to prevent buffer-related defects.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa83"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.full.high.jpg/1726159644184.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.full.high.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.320.high.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.480.high.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.320.medium.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.620.high.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.320.low.jpg/1726159644184.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa83/foregroundimg.img.480.medium.jpg/1726159644184.jpg" alt='Protein electrophoresis troubleshooting, shadow at lane edges' title='tb5'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa83 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa83 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa83 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="pge5"> Shadow at lane edges </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-1960799884"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td>Excess reducing agent in the lysis or sample buffer</td><td>The final concentration of reducing agents for SDS-PAGE should be less than 50 mM for DTT (dithiothreitol) and TCEP (tris(2-carboxyethyl) phosphine), and less than 2.5% for β-ME (β-mercaptoethanol).</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><div class="divider section"><hr /></div><div class="heading section"><h2> General western blot troubleshooting </h2></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5e09"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.full.high.jpg/1726159644249.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.full.high.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.320.high.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.480.high.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.320.medium.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.620.high.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.320.low.jpg/1726159644249.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5e09/foregroundimg.img.480.medium.jpg/1726159644249.jpg" alt='Western blot troubleshooting, nonspecific bands' title='tb6'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5e09 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5e09 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5e09 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="gwb1"> Nonspecific or diffuse bands </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-703667099"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td>Antibody concentration too high</td><td>Reduce concentrations of antibodies, particularly of primary antibody.</td></tr><tr><td>Too much protein loaded on gel</td><td>Reduce the amount of sample loaded on gel.</td></tr><tr><td rowspan="5">Signal from chemiluminescent substrate too strong</td><td>Reduce the length of time the blot is exposed to film.</td></tr><tr><td>Reduce the concentration of the substrate.</td></tr><tr><td>Shorten incubation time of membrane with substrate.</td></tr><tr><td>Completely remove substrate after incubation period.</td></tr><tr><td>Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_44e6"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.full.high.jpg/1726159644304.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.full.high.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.320.high.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.480.high.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.320.medium.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.620.high.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.320.low.jpg/1726159644304.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_44e6/foregroundimg.img.480.medium.jpg/1726159644304.jpg" alt='Western blot troubleshooting, high background' title='tb8'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_44e6 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_44e6 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_44e6 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="gwb2"> High background </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-1630614039"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td>Antibody concentration too high</td><td>Decrease concentration of primary and/or secondary antibody.</td></tr><tr><td rowspan="5">Incompatible blocking buffer</td><td>Do not use milk with avidin–biotin system. Milk contains biotin, which will result in high background.</td></tr><tr><td>When probing for phosphoproteins, avoid phosphate- based buffers like PBS and phosphoprotein-containing blockers like milk or casein. Instead, block with BSA in Tris-buffered saline.</td></tr><tr><td>Test for cross-reactivity in blocking buffer by blocking a clean piece of membrane, incubating with antibodies, and then detecting with the substrate of choice.</td></tr><tr><td>When using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity.</td></tr><tr><td>Try a different blocking buffer. Use our <a href="/sg/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-buffers.html">blocking buffer selection guide</a> to find the most compatible blocking buffer for your experiment.</td></tr><tr><td rowspan="5">Insufficient blocking of nonspecific sites</td><td>Increase the concentration of protein in the blocking buffer.</td></tr><tr><td>Optimize blocking time and/or temperature. Block for at least 1 hour at room temperature (RT) or overnight at 4°C.</td></tr><tr><td>Adding Tween 20 detergent to the blocking buffer can help minimize background. However, too much detergent can interfere with antibody binding. A final concentration of 0.05% often works well. For ease of use, choose a blocking buffer that already contains 0.05% Tween 20 detergent, such as Thermo Scientific StartingBlock T20 Blocking Buffer (<a href="/order/catalog/product/37543">TBS</a>) or (<a href="/order/catalog/product/37539">PBS</a>) or SuperBlock T20 Blocking Buffer (<a href="/order/catalog/product/37536">TBS</a>) or (<a href="/order/catalog/product/37516">PBS</a>).</td></tr><tr><td>Prepare antibody dilutions in a blocking buffer that contains 0.05% Tween 20 detergent.</td></tr><tr><td>Use <a href="/order/catalog/product/46640">Thermo Scientific SuperSignal Western Blot Enhancer</a> to reduce background and enhance detection of low-abundance and weakly immunoreactive antigens.</td></tr><tr><td rowspan="2">Insufficient washing</td><td>Increase the number of washes and/or the volume of buffer used.</td></tr><tr><td>Add Tween 20 detergent to the wash buffer to a final concentration of 0.05%. If the concentration of Tween 20 detergent is too high, it can strip proteins off the membrane.</td></tr><tr><td rowspan="5">Membrane handled improperly</td><td>Wet and activate membrane according to manufacturer’s instructions.</td></tr><tr><td>Always wear clean gloves or use forceps when handling membrane.</td></tr><tr><td>Cover the membrane with liquid at all times to prevent drying.</td></tr><tr><td>Use agitation during all incubations.</td></tr><tr><td>Handle membrane carefully—damage to the membrane can cause nonspecific binding.</td></tr><tr><td rowspan="2">Contamination of equipment or materials</td><td>Prepare fresh buffers and filter them before use.</td></tr><tr><td>Use only clean and contaminant-free electrophoresis equipment, blotting equipment, and incubation trays.</td></tr><tr><td rowspan="5">Signal from chemiluminescent substrate too strong</td><td>Reduce the length of time the blot is exposed to film.</td></tr><tr><td>Reduce the concentration of the substrate.</td></tr><tr><td>Shorten incubation time of membrane with substrate.</td></tr><tr><td>Completely remove substrate after incubation period.</td></tr><tr><td>Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_e285"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.full.high.jpg/1726159644357.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.full.high.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.320.high.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.480.high.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.320.medium.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.620.high.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.320.low.jpg/1726159644357.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_e285/foregroundimg.img.480.medium.jpg/1726159644357.jpg" alt='Western blot troubleshooting, weak or no signal' title='tb9'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_e285 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_e285 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_e285 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="gwb3"> Weak or no signal </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table--1900589736"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="9">Incomplete or inefficient transfer</td><td>After transfer, stain the gel with a total protein stain to determine transfer efficiency.</td></tr><tr><td>After transfer, assess transfer efficiency by staining the membrane with the Thermo Scientific Pierce Reversible Protein Stain Kit (<a href="/order/catalog/product/24585">PVDF</a>) or (<a href="/order/catalog/product/24580">Nitrocellulose membranes</a>). </td></tr><tr><td>Ensure sufficient contact between the gel and membrane during transfer by using a gel roller across the transfer stack.</td></tr><tr><td valign="top"><p>Ensure that the stack is placed in the transfer apparatus in the proper orientation such that proteins will migrate onto the membrane.</p></td></tr><tr><td>Wet and activate the membrane according to the manufacturer’s instructions.</td></tr><tr><td>Use a positive control, such as prestained molecular weight markers, to assess transfer efficiency.</td></tr><tr><td>Use molecular weight markers compatible with a western- imaging substrate, such as the <a href="/order/catalog/product/LC5615">Invitrogen iBright Prestained Protein Ladder</a> or <a href="/order/catalog/product/LC5602">Invitrogen MagicMark XP Western Protein Standard</a>, as a positive control.</td></tr><tr><td>Increase transfer time and/or voltage.</td></tr><tr><td>Make sure sample preparation conditions have not destroyed the antigenicity of the sample. (Some proteins cannot be run under reducing conditions.)</td></tr><tr><td rowspan="5">Insufficient binding to membrane</td><td>For low molecular weight (MW) antigens, add 20% methanol to the transfer buffer to help binding and prevent proteins from passing through membrane.</td></tr><tr><td>Reduce transfer time. Low MW antigens may pass through membrane.</td></tr><tr><td>For high MW antigens, add 0.01–0.05% SDS to transfer buffer to pull proteins from the gel onto membrane.</td></tr><tr><td>Change membrane type (NC vs. PVDF).</td></tr><tr><td>Change to membrane with smaller pore size.</td></tr><tr><td rowspan="2">Antibody concentration too low</td><td>Increase antibody concentrations. Antibody may have poor affinity for the target protein.</td></tr><tr><td>Antibody may have lost activity. Perform a dot blot to determine activity.</td></tr><tr><td>Insufficient antigen present</td><td>Load more protein onto the gel.</td></tr><tr><td rowspan="2">Antigen masked by blocking buffer</td><td>Decrease concentration of protein in blocking buffer.</td></tr><tr><td>Try a different blocking buffer. Use our <a href="/sg/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-buffers.html">blocking buffer selection guide</a> to find the most compatible blocking buffer for your experiment.</td></tr><tr><td>Buffer contains sodium azide</td><td>Sodium azide inhibits HRP. Do not use it with HRP-conjugated antibodies.</td></tr><tr><td rowspan="4">Signal from chemiluminescent substrate too weak</td><td>Increase incubation time of membrane with substrate.</td></tr><tr><td>Increase film exposure time.</td></tr><tr><td>Ensure that the substrate is not expired.</td></tr><tr><td>When you have minimal protein, use <a href="/order/catalog/product/34096">Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate</a> to maximize your western blot signals.</td></tr><tr><td rowspan="2">Membrane has been stripped and reprobed</td><td>Avoid repeated stripping of the same membrane.</td></tr><tr><td>Shorten incubation time in stripping buffer to prevent loss of antigen.</td></tr><tr><td>Digestion of antigen on membrane</td><td>Blocking substance may have proteolytic activity (e.g., gelatin).</td></tr><tr><td>Protein degradation from prolonged blot storage</td><td>Prepare new blot.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><div class="divider section"><hr /></div><div class="heading section"><h2> Fluorescent western blotting troubleshooting </h2></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa79"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.full.high.jpg/1726159644420.jpg' media="(min-width: 1px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.full.high.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.320.high.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.480.high.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.320.medium.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.620.high.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.320.low.jpg/1726159644420.jpg,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_aa79/foregroundimg.img.480.medium.jpg/1726159644420.jpg" alt='Fluorescent western blot troubleshooting nonspecific bands' title='tb10'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa79 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa79 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_aa79 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="fwb1"> Nonspecific or diffuse bands </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-721122183"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="2">Poor antibody specificity for the target of interest</td><td>Evaluate additional primary antibodies.</td></tr><tr><td>Use only primary antibodies validated* for western blots.</td></tr><tr><td>Poor sample integrity</td><td>Sample degradation due to overheating or protease activity results in target breakdown and low target recognition by the antibody. For example, do not boil SDS-PAGE samples in SDS sample buffer, but rather heat them at 70°C for 10 minutes to avoid proteolysis.</td></tr><tr><td rowspan="3">Antibody cross-reactivity in multiplex detection</td><td>Choose primary antibodies raised in distantly related host species.</td></tr><tr><td>Use highly cross-adsorbed secondary antibodies.</td></tr><tr><td>Reduce the amount of the secondary antibody used, to remain within the optimal performance range.</td></tr><tr><td rowspan="3">Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band)</td><td>Avoid spectrally close conjugates, especially when the signal is very strong.</td></tr><tr><td>Ensure that your fluorescent dyes can be distinctly detected on your imager.</td></tr><tr><td>Use the autoexposure feature on the instrument to determine the optimal exposure time for each channel.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt2-c0"><div container-id="cq-colctrl-lt2" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5d80"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_5d80/foregroundimg.img.full.high.jpg/1726159644471.jpg' media="(min-width: 1px)"><img 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alt='Fluorescent western blot troubleshooting, weak or no signal' title='tb11'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5d80 img{ width:100%; max-width: 461px; } </style><script type="text/javascript"> $( document ).ready(function() { // apply fillIt plugin var fillClasses = "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5d80 .image-contain" + "," + "#cq-image-jsp-"+"e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_5d80 .image-cover" ; // Due to the issue explained below, we are binding 'one' instead of 'on' and we need it only once. $(fillClasses).load(function() { $(this).fillIt(); }); // Force load image to avoid cache issue, a bug which makes image reset height when having more than one image/image-text components on the same page and rendering after edit. $(fillClasses).each(function(){ if(this.complete){ $(this).load(); } }); if(!!window.picturefill){ window.picturefill(); } }); </script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="fwb2"> Weak or no signal </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table--873945557"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="5">Poor antibody specificity for the target of interest</td><td>Increase primary antibody concentration.</td></tr><tr><td>Ensure primary antibody has a good titer and is specific for the antigen to be detected.</td></tr><tr><td>For a low-abundance target in a cell or tissue lysate, increase the amount of primary antibody or the amount of sample loaded on the gel.</td></tr><tr><td>Extend the incubation time to overnight at 4°C, or 3–6 hours at room temperature.</td></tr><tr><td>Try using an antibody enhancer.</td></tr><tr><td rowspan="3">Lost activity of antibody</td><td>Ensure the antibody was stored appropriately.</td></tr><tr><td>Check the expiration date of the antibody.</td></tr><tr><td>Avoid multiple uses of prediluted antibodies.</td></tr><tr><td rowspan="2">Imaging exposure time is too short</td><td>Increase exposure time.</td></tr><tr><td>Utilize the Smart Exposure feature to obtain an optimal image on the iBright FL1000 system.</td></tr><tr><td>Incorrect instrument settings</td><td>Ensure the correct excitation and emission ranges are selected for the intended fluorophore.</td></tr><tr><td>Use of detergent</td><td>Too much detergent or the nature of the detergent can result in washing away the signal—decrease or eliminate detergent.</td></tr><tr><td rowspan="3">Blocking buffer blocks antigen</td><td>Some blocking solutions can mask the blot and reduce the availability of the antigen to the antibody, especially if the blocking step is >1 hour.</td></tr><tr><td>Dilute the primary antibody in wash buffer.</td></tr><tr><td>Evaluate another blocking buffer.</td></tr><tr><td rowspan="3">Quantity of sample loaded on the gel</td><td>Too much lysate can overcrowd your specific target and reduce the antibody sensitivity.</td></tr><tr><td>Too little lysate leads to insufficient availability of the target of interest.</td></tr><tr><td>Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load.</td></tr><tr><td rowspan="2">Poor transfer of protein, or loss of the protein after transfer</td><td>Check transfer conditions to confirm protein transfer.</td></tr><tr><td>Reoptimization may be required when probing for a new protein.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt2"><!-- Col -- START --><div 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</script></div></div></div></div><!-- Col -- BREAK --><div class="parsys_column cq-colctrl-lt2-c1"><div container-id="cq-colctrl-lt2" comp-id="B1"><div class="heading section"><h3 id="fwb3"> Background issues (high, uneven, or speckled) </h3></div></div></div><!-- Col -- END --></div><!-- ColCtrl -- END --><div style="clear:both"></div><div class="text parbase section"><div class="table-component-wrapper"><table class="rte-table" id="table-1138395628"><tbody><tr><td width="25%"><b>Possible cause</b></td><td><b>Solutions</b></td></tr><tr><td rowspan="3">High background due to membrane contamination</td><td>Handle the membrane using clean forceps and clean incubation trays or dishes.</td></tr><tr><td>Determine the best blocking buffer for your application— primary antibodies will react differently in different blocking buffers. Blocking buffers like normal animal sera or milk may result in cross-reactivity.</td></tr><tr><td>Limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and actually increase nonspecific background. After blocking, detergents can be used.</td></tr><tr><td>Artifacts from overloading the protein marker or ladder</td><td>Load less of the molecular weight marker onto the gel.</td></tr><tr><td rowspan="3">Nonoptimal wash or diluent solutions</td><td>Use a wash buffer with 0.1–0.2% Tween 20 detergent.</td></tr><tr><td>Prepare the secondary antibody dilution with 0.05% Tween 20 detergent.</td></tr><tr><td>Increase the number or duration of wash steps.</td></tr><tr><td>High background from an excess of secondary antibody</td><td>Optimize the secondary antibody dilution depending on the dye being used, following the vendor-recommended dilution and adapting accordingly.</td></tr><tr><td rowspan="2">Blotchy or uneven background due to the membrane drying out</td><td>Ensure good coverage of the whole blot during all incubation steps.</td></tr><tr><td>Ensure consistent agitation during every incubation step.</td></tr><tr><td>Incorrect choice of membrane</td><td>The nature of the membrane can affect the background; for example, PVDF membranes can autofluoresce and cause high background, so use low-fluorescence PVDF membranes.</td></tr><tr><td rowspan="4">Speckles and fingerprints on the membrane</td><td>Use clean forceps to handle the membrane and avoid directly touching the membrane; particulates and contaminants from unclean tools may fluoresce.</td></tr><tr><td>Use clean incubation trays or dishes—rinsing with methanol followed by water will help dissolve residual dried dyes from previous uses.</td></tr><tr><td>Clean transfer devices and dusty consumables if using a wet transfer method, as they can introduce speckles.</td></tr><tr><td>Clean the imager surface with ethanol to remove dust, lint, and residue before capturing the image.</td></tr></tbody></table></div></div><div class="text parbase section"><p style="text-align: right;"><a href="#top">(Top)</a></p></div><!-- ColCtrl -- START --><div comp-order="bycol" class="parsys_column cq-colctrl-lt3"><!-- Col -- START --><div class="parsys_column cq-colctrl-lt3-c0"><div container-id="cq-colctrl-lt3" comp-id="A1"><div class="image parbase section"><div id="cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_8814"><a href="/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information.html"><picture><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.320.high.png/1726159644567.png' media="(min-width: 1px)"><source srcset='https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.480.high.png/1726159644567.png' media="(min-width: 490px)"><img srcset="https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.full.high.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.320.high.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.480.high.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.320.medium.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.620.high.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.320.low.png/1726159644567.png,https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting/jcr:content/MainParsys/image_8814/foregroundimg.img.480.medium.png/1726159644567.png" alt='Education homepage' title='Education homepage'></picture><style> #cq-image-jsp-e8121e56-e0f9-488f-b487-83e1a9e8c65cMainParsysimage_8814 img{ width:100%; 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