Practical Tips for Chemiluminescent Western Blots

Practical Tips for Chemiluminescent Western Blots

<div class="level1"> Here are some practical tips from Advansta for every step of your chemiluminescent Western blot. </div> <!--more--> <h2 id="tissue_preparation" class="sectionedit2">Tissue Preparation</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Make sure <a href="https://advansta.com/wikis/preparing-samples-for-western-blot-analysis-cell-lysis/" target="_blank">samples are completely lysed</a> to maximize recovery of proteins.</div></li> <li class="level1"> <div class="li"><a href="https://advansta.com/chill-isolating-proteins/" target="_blank">Keep samples cold</a> to prevent protein degradation.</div></li> <li class="level1"> <div class="li">Use <a href="https://advansta.com/oh-protein-go-proteases-protease-inhibitors/" target="_blank">protease inhibitors</a> in lysis buffer to prevent degradation of the sample.</div></li> <li class="level1"> <div class="li">Determine the <a href="https://advansta.com/wikis/preparing-samples-for-western-blot-analysis-protein-quantification/" target="_blank">protein concentration</a> of each sample to calculate the amount of sample to load on the gel.</div></li> <li class="level1"> <div class="li">When freezing samples, make small aliquots to prevent multiple freeze-thaws.</div></li> </ul> </div> <h2 id="electrophoresis" class="sectionedit3">Electrophoresis</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Make sure the gel has no imperfections and is properly set before using.</div></li> <li class="level1"> <div class="li"><a href="https://advansta.com/ask-advansta-why-do-we-heat-protein-samples-before-loading/" target="_blank">Heat samples prior to loading</a> to break secondary structures. Use a reducing agent when required.</div></li> <li class="level1"> <div class="li">Make sure the well size is appropriate for the sample volume to prevent carryover of samples.</div></li> <li class="level1"> <div class="li">Do not overload the lane with too much protein; it will cause distortion of the lane.</div></li> <li class="level1"> <div class="li">Run the gel at the correct voltage: running the gel at a voltage that is too high can cause fuzzy bands or “smiling” bands.</div></li> </ul> </div> <h2 id="transfer" class="sectionedit4">Transfer</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Handle the membrane with gloved hands to prevent marks on the membrane.</div></li> <li class="level1"> <div class="li">Use “powder-free” gloves. Particles in powdered gloves can stick to membranes and cause <a href="https://advansta.com/wikis/high-background-in-western-blots-causes-and-solutions-2/" target="_blank">high background</a>.</div></li> <li class="level1"> <div class="li">Wash gloved hands before handling membranes and minimize direct contact with working areas. Even non-powdered gloves can contain a tiny amount of residual process chemicals that stick to membranes and cause background or other artifacts.</div></li> <li class="level1"> <div class="li">Wash all transfer equipment including transfer pads to reduce background.</div></li> <li class="level1"> <div class="li">Make sure <a href="https://advansta.com/membrane-matters/" target="_blank">PVDF membrane</a> is pre-wetted with methanol.</div></li> <li class="level1"> <div class="li">Remove all air bubbles when setting up the transfer sandwich to prevent incomplete transfer or fuzzy bands.</div></li> <li class="level1"> <div class="li"><a href="https://advansta.com/wikis/western-blotting-transfer-2/" target="_blank">Optimize the transfer conditions</a>.</div> <ul> <li class="level2"> <div class="li">Alter the concentration of SDS and methanol in transfer buffer to increase transfer efficiency.</div></li> <li class="level2"> <div class="li">Decrease transfer time for smaller proteins, increase transfer time for larger proteins.</div></li> <li class="level2"> <div class="li">Use a high performance transfer buffer such as<span class="Apple-converted-space"> </span><a href="https://advansta.com/products/FLASHBlot_Transfer_Buffer/" target="_blank">FLASHBlot</a><span class="Apple-converted-space"> </span>to enhance transfer efficiency and increase protein retention on the membrane.</div></li> </ul> </li> <li class="level1"> <div class="li">Chill transfer setup if necessary to prevent overheating.</div></li> <li class="level1"> <div class="li">Use prestained molecular weight markers to monitor transfer.</div></li> <li class="level1"> <div class="li">Use<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/AdvanStain_Ponceau/?root=356/" href="https://advansta.com/products/AdvanStain_Ponceau/" target="_blank" rel="nofollow">AdvanStain Ponceau</a><span class="Apple-converted-space"> </span>or a similar stain to visualize transfer efficiency.</div></li> </ul> </div> <h2 id="blocking" class="sectionedit5">Blocking</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Prepare <a href="https://advansta.com/wikis/western-blotting-blocking-buffers-2/" target="_blank">blocking buffer </a>fresh just before use to prevent contamination and high background.</div></li> <li class="level1"> <div class="li">Filter the blocking solution to prevent clumps that will stick to the membrane causing speckles and blotches.</div></li> <li class="level1"> <div class="li">Completely solubilize detergent in the blocking buffer prior to use to prevent high background.</div></li> <li class="level1"> <div class="li">Optimize the blocking agent to reduce background and increase signal.</div> <ul> <li class="level2"> <div class="li">Be aware that excessive blocking can mask some antigens. Use 1% milk rather than 5%.</div></li> <li class="level2"> <div class="li">Try different blocking agents to reduce background.</div> <ul> <li class="level3"> <div class="li">Primary antibodies can recognize components of blocking agents causing high background.</div></li> <li class="level3"> <div class="li">Some detection reagents cross-react with blocking solutions.</div></li> </ul> </li> <li class="level2"> <div class="li">Use a protein free blocking agent such as<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/AdvanBlock-PF/?root=294/" href="https://advansta.com/products/AdvanBlock-PF/" target="_blank" rel="nofollow">AdvanBlock-PF</a>.</div></li> </ul> </li> <li class="level1"> <div class="li">Use a TBS-based buffer system when detecting phospho-proteins.</div></li> <li class="level1"> <div class="li">Do not use milk-based blockers or casein if you plan to use any biotinylated antibody and streptavidin detectors. Milk contains biotin and will cause high background when combined with these reagents.</div></li> </ul> </div> <h2 id="incubations_and_washes" class="sectionedit6">Incubations and Washes</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Keep all containers and solutions free of dirt to prevent background on blot.</div></li> <li class="level1"> <div class="li"><a href="https://advansta.com/products/Incubation_trays/" target="_blank">Cover containers</a> when incubating or washing blot to prevent particulates from falling onto the blot.</div></li> <li class="level1"> <div class="li">Use a shaker to prevent uneven distribution of buffers.</div></li> <li class="level1"> <div class="li">Increase wash time to decrease high background.</div></li> <li class="level1"> <div class="li">Decrease wash time to increase signal.</div></li> </ul> </div> <h2 id="antibodies" class="sectionedit7">Antibodies</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Make sure the <a href="https://advansta.com/choose-good-primary-antibody/" target="_blank">primary antibody</a> can detect the protein of interest under the conditions used (i.e. some antibodies only recognize tertiary structures in native proteins).</div></li> <li class="level1"> <div class="li"><a href="https://advansta.com/wikis/protocol-dot-blot-checkerboard-titration-of-antibodies-2/" target="_blank">Titrate primary and secondary antibodies</a> to find the optimum concentrations for each experiment.</div></li> <li class="level1"> <div class="li">To decrease general background, dilute the secondary antibody further.</div></li> <li class="level1"> <div class="li">To decrease <a href="https://advansta.com/wikis/multiple-bands-in-western-blots-causes-and-solutions-2/" target="_blank">multiple bands</a> that are non-specific, dilute the primary antibody further.</div></li> <li class="level1"> <div class="li">Use positive and negative control samples to make sure the antibody is detecting the right target.</div></li> <li class="level1"> <div class="li">Centrifuge antibody aliquots prior to removing sample to avoid aggregates that can cause patches or splotches on the blot.</div></li> <li class="level1"> <div class="li">Optimize the primary antibody incubation time. While a 1-2 hour incubation at room temperature works well for most antibodies, some antibodies require an overnight incubation at 4°C for optimum performance.</div></li> </ul> </div> <h2 id="detection" class="sectionedit8">Detection</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">If refrigerated, bring the substrate to room temperature prior to use for optimum enzyme activity.</div></li> <li class="level1"> <div class="li">If using<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/WesternBright_ECL/?root=293/" href="https://advansta.com/products/WesternBright_ECL/" target="_blank" rel="nofollow">Advansta's WesternBright substrates</a>, always keep them at room temperature. Storing these substrates in a refrigerator can reduce their shelf life.</div></li> <li class="level1"> <div class="li">Make sure all wash buffer is sufficiently drained from the membrane prior to incubation with the substrate. The pH of the substrate is different than the pH of the wash buffer. If the substrate is diluted with wash buffer, then the pH change can shorten the signal duration.</div></li> <li class="level1"> <div class="li">Always cover the blot with sufficient substrate; 0.1 ml of substrate per 1 cm²<span class="Apple-converted-space"> </span>of membrane is recommended.</div></li> <li class="level1"> <div class="li">Don't let the membrane dry out after adding substrate. Cover it with saran wrap or<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/Development_Folders/?root=293/" href="https://advansta.com/products/Development_Folders/" target="_blank" rel="nofollow">plastic covers</a>.</div></li> <li class="level1"> <div class="li">After adding substrate to the membrane, wait 2-5 minutes (following the manufacturer’s recommendations) for the reaction to reach peak light generation.</div></li> <li class="level1"> <div class="li">To troubleshoot substrate issues, use a positive control for substrate development such as Advansta's<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/WesternBright_ChemiPen/?root=293/" href="https://advansta.com/products/WesternBright_ChemiPen/" target="_blank" rel="nofollow">WesternBright ChemiPen</a>.</div></li> <li class="level1"> <div class="li">Don’t strip and reuse the blot. Stripping removes protein from the blot.</div></li> </ul> Photo courtesy of <a href="https://www.flickr.com/photos/anneh632/5262162215/in/photolist-91ZXuK-9cmR5u-bgW2nX-raXM86-kpzUn2-bmfPwd-bgVxVp-r8EMCw-4CCLSQ-r65Qgf-bgVwqR-qTvtk4-raPiMB-hYiN1d-bgVNcT-4CCizy-bgVXGt-9cmScQ-dfX4Ac-qrwES-7RzzXj-ngYv1V-rmNJXM-63CNax-qTwVGD-raPqon-qTvc1R-6iW2Qy-aXRMYK-qJUZWa-bgVnVv-bjXh6i-raNRh2-raXWyR-dC4FZT-rLSRBP-6hXV3e-aNU2Sx-qTuV3Z-5ZmxAK-r8EeRL-9193D9-adwcq6-8TJfJ8-e1xJhL-qn9Kod-pZ1D4w-eaiQcw-nu91FH-tLxgBz" target="_blank">Anne Swoboda</a>. </div>