TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>This protocol describes a method to determine optimal blocking conditions for NIR Western blot detection with the Odyssey family of Imagers.</p>
<p> </p>
<p>It is for use with the Millipore Immobilon®-FL PVDF membrane.</p>
</div>
</div>
</div>

[GUIDELINES]
<p><strong>I. Introduction</strong></p>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>This document describes a method to determine optimal blocking conditions for NIR Western blot detection with the Odyssey family of Imagers. The specific lysate and antibodies used in your system will be evaluated in four different blocking buffer systems. The buffering system will also be evaluated in this experiment. Odyssey Blocking Buffer is provided in either TBS (TRIS Buffered Saline) or PBS (Phosphate Buffered Saline) formulation. In general, the washing step should in- clude the same buffering system as the blocking step (e.g., 1X TBS wash when using Odyssey Blocking Buffer (TBS)).</p>
<p> </p>
<p><strong>II. Required Reagents</strong></p>
<p> </p>
<table style="width: 758px; height: 681px;">
<tbody>
<tr>
<td style="width: 499px;"> </td>
<td style="width: 247px;">LI-COR P/N</td>
</tr>
<tr>
<td style="width: 499px;">Odyssey Protein Molecular Weight Marker</td>
<td style="width: 247px;">928-40000</td>
</tr>
<tr>
<td style="width: 499px;">IRDye® Secondary Antibodies (LI-COR)</td>
<td style="width: 247px;"><a href="https://www.licor.com/bio/products/reagents/secondary_antibodies/" target="_blank">various</a></td>
</tr>
<tr>
<td style="width: 499px;">Blocking Buffer</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<ul>
<li>Blocking Buffer Optimization Kit</li>
</ul>
</td>
<td style="width: 247px;">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>927-40040</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 499px;">          -  Odyssey Blocking Buffer (TBS)</td>
<td style="width: 247px;">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>927-50000; 927-50100</p>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 499px;">          -  Odyssey Blocking Buffer (PBS)</td>
<td style="width: 247px;">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>927-40000; 927-40100</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 499px;">          -  Casein Blocking Buffer (PBS)</td>
<td style="width: 247px;"> 927-40200;927-40300</td>
</tr>
<tr>
<td style="width: 499px;">
<ul>
<li>Blocking Buffer of your choice (milk, BSA, etc.)</li>
</ul>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">Membrane</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<ul>
<li>Odyssey Nitrocellulose (0.22 µm)</li>
</ul>
</td>
<td style="width: 247px;">
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>926-31090; 926-31092</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td style="width: 499px;">
<ul>
<li>4X Protein Sample Loading Buffer and PVDF Membrane Kit</li>
</ul>
</td>
<td style="width: 247px;">926-31097</td>
</tr>
<tr>
<td style="width: 499px;">Primary Antibodies (primary antibodies must be from host species compatible with the secondary antibodies being used. If using subclass-specific antibodies, please refer to Technical Note 'Western Blot and In-Cell Western™ Assay Detection Using IRDye® Sub-class Specific Antibodies').</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Tween® 20</p>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>PBS Buffer (1X)</p>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>TBS Buffer (1X)</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Methanol (when using Immobilon®-FL PVDF membrane)</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>SDS (when using Immobilon-FL PVDF membrane)</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Western Blot Incubation Box</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> 929-97201; 929-97205; 929-97210</td>
</tr>
<tr>
<td style="width: 499px;">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Aerius or Odyssey Family Imager</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</td>
<td style="width: 247px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>III. Gel Preparation for Blocker Optimization</strong></p>
<p> </p>
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Standard protein electrophoresis conditions and reagents can be used for gel and sample preparation. Following is a suggested template for sample electrophoresis to maximize blocker optimization and efficiently choose the best blocking conditions for a given primary antibody.</p>
<p> </p>
<p>Using two 15-well gels, load the following samples in the order indicated:</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g7sc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g7sc2w6.png" data-ofn="Screen Shot 2016-12-13 at 10.20.44 AM.png" /></p>
<p> </p>
<p><strong>IV. Western Blocker Optimization Method</strong></p>
<p> </p>
<div class="page" title="Page 4">
<div class="layoutArea">
<div class="column">
<p>Western blots should be prepared using standard blotting procedures and Millipore Immobilon®-FL PVDF or Odyssey Nitrocellulose Membrane. Allow blots to dry for at least 1 hour before proceeding with detection. Dry blots can be stored between filter paper overnight at room temperature.</p>
<p> </p>
<p><em>NOTE: Membranes should be handled only by their edges, with clean forceps. Take great care to never touch the membrane with bare or gloved hands.</em></p>
<p> </p>
<p><em>NOTE: Do not write on membranes with an ink pen or marker, as the ink will fluoresce on the Odyssey Imager. Mark with pencil or Odyssey Pen (P/N 926-71804) to avoid this problem. Use pencil only for PVDF membrane, because wetting in methanol will cause ink to run.</em></p>
<p> </p>
<p>If using the gel configuration described in Section III (Gel Preparation for Blocker Optimization), cut each membrane along the protein marker in lane 8 as shown in Figure 1. Be careful not to touch the membranes with bare or gloved hands. Label appropriately with pencil.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g7tc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g7tc2w6.png" data-ofn="Screen Shot 2016-12-13 at 10.22.20 AM.png" /></p>
<p> </p>
<p>After cutting membranes, proceed to 'STEPS'</p>
<p> </p>
<p><strong>Imaging Steps Continued: </strong></p>
<p> </p>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">11. Visual inspection of images with Image Studio software or Odyssey application software can be used to determine which blocking buffer works best for the primary antibody you are testing.</div>
<div class="column">
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<ul>
<li>View all blots together in a single image, with uniform image display settings, to compare membrane background levels and band intensity (Fig. 2A).</li>
<li>Individually adjust the image display settings for each blot to get the “best” image (Fig. 2B).</li>
<li>Evaluate non-specific banding in each blocking buffer condition.</li>
<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.</li>
<li>Trade-offs 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>
</ul>
<p> </p>
<p>12. Quantitative analysis of specific bands on each blot (Section V; Fig. 2) can be used to determine if signal intensity (after background subtraction) is significantly different between blocker types.</p>
<p> </p>
<p><strong>V. Example: Analysis of Optimization Blots</strong></p>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<ol>
<li>
<p>In this optimization experiment, ERK1/2 was detected in serial dilutions of A431 cell lysates.</p>
</li>
<li>
<p>After imaging, blocking buffer performance was evaluated. In addition to visual comparison, quantitative analysis was used to examine signal intensity of the ERK1/2 doublet under the conditions tested.</p>
</li>
</ol>
<p> </p>
<p><strong>Figure 2A.</strong> All blots were viewed together in a single image, with identical image display settings. Signal intensity values are shown in each lane. Higher membrane background was observed on the blot blocked with 5% milk in TBS.</p>
<p> </p>
<div class="page" title="Page 8">
<div class="layoutArea">
<div class="column">
<p><strong>Figure 2B.</strong> After cropping, image display settings were adjusted individually to display the “best” image for each blot. Signal intensity values are shown in each lane.</p>
<p> </p>
<div class="page" title="Page 8">
<div class="layoutArea">
<div class="column">
<p><strong>Figure 2C.</strong> Quantification data from all four blots were plotted. Band intensity was highest in Odyssey Blocking Buffer (PBS). Intensity was lowest in 5% milk and Odyssey Blocking Buffer (TBS).</p>
<p> </p>
<div class="page" title="Page 8">
<div class="layoutArea">
<div class="column">
<p><strong>Signal intensity values in Figures 2A and 2B were not affected by changes to the image display settings.</strong> <em>Signal intensity measurements are derived from the raw image data. Image display settings adjust the visual mapping of raw data to the pixels on the computer monitor, but do not affect the raw data.</em></p>
<p> </p>
<p>     3. Analysis and interpretation of optimization data</p>
<div class="page" title="Page 8">
<div class="layoutArea">
<div class="column">
<ul>
<li>Blocking with 5% milk in TBS resulted in higher membrane background than other blocking conditions (Fig. 2A). For comparison of membrane background levels, blots were viewed together as a single image, with uniform image display settings.</li>
<li>The strongest signals were observed with Odyssey Blocking Buffer (PBS) (OBB-PBS, Fig. 2C). Signals observed with casein blocker were also good.</li>
<li>Weaker signals were observed with Odyssey Blocking Buffer (TBS) (OBB-TBS) and 5% milk.</li>
<li>Non-specific background bands were seen with OBB (TBS), OBB (PBS), and Casein blocking buffers (Fig 2B, arrows). Non-specific banding was reduced with 5% milk (TBS).</li>
<li>OBB (PBS) and Casein may be appropriate choices for this antibody-antigen pair.</li>
<li>If non-specific banding is a concern, 5% milk (TBS) could be used with this primary antibody. Band intensity would be somewhat reduced, but strong bands would still be visible. However, increased membrane background may make it difficult to detect fainter bands. Optimization of other factors, such as antibody dilution, may also reduce non-specific banding.</li>
<li><strong>The “best” choice of blocking buffer depends on the antigen-antibody pair you are using. Choose the blocking conditions that are most appropriate for the context and goals of your experiment.</strong></li>
</ul>
<p> </p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g7uc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g7uc2w6.png" data-ofn="Screen Shot 2016-12-13 at 10.55.39 AM.png" /></strong></p>
<p> </p>
<div class="page" title="Page 9">
<div class="layoutArea">
<div class="column">
<p><strong>Figure 2.</strong> Two-fold serial dilutions of A431 cell lysate were separated by elec- trophoresis (sample loads ranged from 313 ng to 10 μg, left to right). Primary antibody was loaded in the first lane of each gel as a positive control. Gels were transferred to nitrocellulose membrane and incubated in the designated buffer for blocking. Membranes were probed with rabbit anti-ERK1/2 (Santa Cruz Biotechnology; sc-94), and then IRDye® 680RD goat anti-rabbit IgG (LI-COR; P/N 926- 68071) prior to imaging on Odyssey CLx.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g7vc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g7vc2w6.png" data-ofn="Screen Shot 2016-12-13 at 10.56.23 AM.png" /></p>
<p> </p>
<p><strong>Tips</strong></p>
<ul>
<li>Follow the protocol carefully.</li>
<li>For additional Odyssey Western detection tips, visit <a href="http://www.licor.com/WesternBlotTips" target="_blank">www.licor.com/WesternBlotTips</a></li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.