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<p>The independent channels of the LI-COR MPX (Multiplex) Blotter make it possible to optimize blocking buffer, primary antibody dilution, and secondary antibody dilution on a single Western blot. Western blotting procedures that generate a blot of 7.0 x 8.5 cm are easily adapted to the MPX format. The process fits into any laboratory’s standard Western blot workflow. Both home- made and pre-cast gels can be used to generate blots.</p>
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<p>Electrophoresis and transfer to nitrocellulose membrane are performed under standard conditions. Clamping the blot into the MPX Blotter creates up to 24 independent channels, allowing different conditions to be tested in each channel. The range of usable channels per sample is relative to comb size. For Western blot optimization, a single-well gel (“prep gel”) is all that is needed. For this application, any detection method can be used, including near-infrared (NIR) fluorescence and chemiluminescence. This document presents general guidelines for use with the Odyssey® family of Infrared Imaging Systems.</p>
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[BEFORE_START]
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<p>Developed for:</p>
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<p><strong>Aerius, and Odyssey® Family of Imaging Systems</strong></p>
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<p>Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model </p>
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[GUIDELINES]
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<p><strong>Gel Preparation</strong></p>
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<li>A wide variety of gel matrices are compatible with the MPX Blotter system.If you are pouring your own gels, your gel casting system can be used with a single-well comb such as the LI-COR Single Marker/One Lane Comb (921-00200, 1 mm thickness). Alternatively, pre-cast gels can be purchased and used. Table 1 lists several suggested types of pre-cast gels, and indicates the number of usable ports that each gel will provide for use with the MPX Blotter.</li>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/x6tesfe.png" width="634" height="151" data-src="https://s3.amazonaws.com/pr-journal/x6sesfe.png" data-ofn="Screen Shot 2018-05-21 at 4.45.31 PM.png" /></p>
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<p><strong>Molecular Weight Marker</strong></p>
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<li>It is important to have a molecular weight marker that is visible to the eye because the marker is the primary tool used to align the blot in the MPX Blotter. Odyssey® One-Color Marker or Chameleon® Duo Protein Ladder is recommended.</li>
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<p><strong>Electrophoresis</strong></p>
<p><br /> <strong>           IMPORTANT:</strong> The maximum length of the separating gel should not exceed 50 mm—the</p>
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<p>           length of the channels on the MPX Blotter.</p>
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<p><strong>Alignment in MPX Blotter</strong></p>
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<li>Detailed instructions for use of the <a href="http://www.licor.com/mpxuserguide" target="_blank" rel="noopener noreferrer">MPX Blotter</a> are found in the MPX Blotter Multiplex Western Blotting Accessory User Guide.</li>
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<p><strong>Imaging</strong></p>
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<li> Visual inspection of images with Image StudioTM software or Odyssey application software can be   used  to determine which blocking buffer works best for the primary antibody you are testing.</li>
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<p> View all blots together in a single image, with uniform image display settings, to com-</p>
<p> pare membrane background levels and band intensity.</p>
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<p> Individually adjust the image display settings for each blot to get the “best” image.</p>
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<p> Evaluate non-specific banding in each blocking buffer condition.</p>
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<li> Look for blocking buffer conditions that provide strong signals for the expected band(s), low membrane   background, and few non-specific background bands from the primary antibody.<br /> • Tradeoffs may be necessary. Blocking conditions that yield very strong bands might also have higher      membrane background or non-specific banding.</li>
<li>The “best” blocking conditions depend on the antigen-antibody pair you are using. Some primary   antibodies are dramatically affected by blocking conditions. An inappropriate blocker can alter binding   specificity, affecting the intensity of target bands and increasing non-specific banding. The pattern of   non-specific bands may also be affected.</li>
<li> Choose the blocking conditions that are most appropriate for the context and goals of your experiment.</li>
<li> Quantitative analysis of specific bands on each blot will indicate if signal intensity (after background   subtraction) is significantly different between blocker types.</li>
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