Practical Tips for Fluorescent Western Blots

Practical Tips for Fluorescent Western Blots

<div class="level1"> Here are some practical tips from Advansta for every step of your fluorescent 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 samples are completely lysed to maximize recovery of proteins.</div></li> <li class="level3"> <div class="li">Keep samples cold to prevent protein degradation.</div></li> <li class="level3"> <div class="li">Use protease inhibitors in lysis buffer to prevent degradation of the sample.</div></li> <li class="level3"> <div class="li">Determine the protein concentration of each sample to calculate the amount of sample to load on the gel.</div></li> <li class="level3"> <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="level3"> <div class="li">Heat samples prior to loading to break secondary structures. Use a reducing agent when required.</div></li> <li class="level3"> <div class="li">Make sure the well size is appropriate for the sample volume to prevent carryover of samples.</div></li> <li class="level3"> <div class="li">Do not overload the lane with too much protein; it will cause distortion of the lane.</div></li> <li class="level3"> <div class="li">If using fluorescent molecular weight markers, skip a lane before loading test samples in case the signal from the markers is intense.</div></li> <li class="level3"> <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> <li class="level3"> <div class="li">Avoid the use of bromophenol blue which autofluoresces and can cause high background.</div></li> </ul> </div> <h2 id="transfer" class="sectionedit4">Transfer</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Use a<span class="Apple-converted-space"> </span><a href="https://advansta.com/products/Transfer_membranes/" target="_blank">low autofluorescence PVDF membrane</a><span class="Apple-converted-space"> </span>to prevent high background.</div></li> <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 high background.</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 PVDF membrane 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">Optimize the transfer conditions.</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<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/AdvanStain_Scarlet/?root=356/" href="https://advansta.com/products/AdvanStain_Scarlet/" target="_blank" rel="nofollow">a fluorescent stain</a><span class="Apple-converted-space"> </span>to monitor transfer.</div></li> <li class="level1"> <div class="li">Use a pencil to mark the blot as some inks autofluoresce.</div></li> </ul> </div> <h2 id="blocking" class="sectionedit5">Blocking</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Prepare blocking buffer 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> <li class="level3"> <div class="li">Try a blocking agent optimized for fluorescent detection, 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> </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="level3"> <div class="li">Cover containers when incubating or washing blot to prevent particulates from falling onto the blot.</div></li> <li class="level3"> <div class="li">Use a shaker to prevent uneven distribution of buffers.</div></li> <li class="level3"> <div class="li">Increase wash time to decrease high background.</div></li> <li class="level3"> <div class="li">Decrease wash time to increase signal.</div></li> <li class="level3"> <div class="li">Photobleaching is not a concern during incubations and washes – perform these steps on the bench.</div></li> </ul> </div> <h2 id="antibodies" class="sectionedit7">Antibodies</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Store fluorescent antibodies in a light-protected box.</div></li> <li class="level1"> <div class="li">Make sure the primary antibody 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 antibodies</a> to yield highest signal-to-noise ratio. Fluorescent Westerns often require different antibody concentrations than chemiluminescent Westerns.</div> <ul> <li class="level2"> <div class="li">Primary antibodies may need to be increased 2-5 fold.</div></li> <li class="level2"> <div class="li">Secondary antibodies may also need to be increased (1:5000 recommended starting dilution.)</div></li> </ul> </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 multiple bands 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="special_considerations_for_multiplexing" class="sectionedit8">Special Considerations for Multiplexing</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Use multiplexing to detect multiple proteins simultaneously:</div> <ul> <li class="level2"> <div class="li">Perform in-lane normalization controls by simultaneously detecting the loading control</div></li> <li class="level2"> <div class="li">Avoid stripping and reprobing or running multiple blots.</div></li> <li class="level2"> <div class="li">Detect proteins of similar molecular weight.</div></li> <li class="level2"> <div class="li">Quantify post-translational modifications.</div></li> </ul> </li> <li class="level1"> <div class="li">Use primary antibodies made in different species to prevent cross reactivity.</div></li> <li class="level1"> <div class="li">Use secondary antibodies that are highly cross-adsorbed.</div></li> <li class="level1"> <div class="li">Use a<span class="Apple-converted-space"> </span><a class="urlextern" title="http://advansta.com/WesternBright_MCF/?root=294/" href="https://advansta.com/products/WesternBright_MCF/" target="_blank" rel="nofollow">a kit designed for multiplexing.</a></div></li> <li class="level1"> <div class="li">Use fluorophores with optically distinct spectra to avoid spectral overlap.</div></li> <li class="level1"> <div class="li">Always optimize detection of each target separately prior to simultaneous detection.</div></li> <li class="level1"> <div class="li">Detect strong targets in the blue or green channel and weaker targets in the red channel to enhance visualization.</div></li> </ul> </div> <h2 id="detection" class="sectionedit9">Detection</h2> <div class="level2"> <ul> <li class="level1"> <div class="li">Make sure detection equipment is compatible with the fluorescent dyes for both excitation and emission wavelengths.</div></li> <li class="level3"> <div class="li">Clean imaging surface regularly to prevent contamination.</div></li> <li class="level3"> <div class="li">Protect archived blots from light.</div></li> </ul> Photo courtesy of <a class="owner-name truncate" title="Go to Danel Solabarrieta's photostream" href="https://www.flickr.com/photos/danelu/3281839742/in/photolist-611hBQ-66KpGV-5eHmSP-5bqrAM-csvCQh-qTpbi1-7yJzux-7kfQYf-qYhU1t-6qzLFP-cTZ52y-b1aXtZ-7XwjeJ-5VWvoy-bjXjvB-8tFdLL-6LKj4C-bjYjE6-bgVid2-cbhr5S-bgVttX-5wL9PU-bgVWdP-bgVmop-bgVuXT-4ZoprL-bgVpmZ-62UBcL-bgVZig-6LFa6P-6LKiRu-shUY7B-g1smbc-6LKiXC-eKM3mQ-6LFanD-dcZ6qZ-nPN9xv-6LFa1M-6uJ2M6-r55RA6-aoEaKA-fLXNct-roWeqG-s3J4jr-bKX2sZ-fM5Kgp-fM5wbH-833vSQ-rpM713" target="_blank" data-rapid_p="58" data-track="attributionNameClick">Danel Solabarrieta</a>. </div>