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[DESCRIPTION]
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<p>Housekeeping proteins (HKPs) are routinely used for Western blot normalization. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. However, expression of several HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression will introduce error and may alter data analysis and interpretation.</p>
<p> </p>
<p>Before using a housekeeping protein for Western blot normalization, it is critical to validate that its expression is constant across all samples and unaffected by the specific experimental context and conditions.</p>
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<p>This protocol explains how to validate an HKP for use as an internal loading control, by demonstrating that HKP expression is stable in the relevant experimental samples.</p>
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<p><strong>Note:</strong> This protocol is intended for use with near-infrared fluorescent Western blots.</p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
<p> </p>
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<p>In quantitative Western blotting (QWB), normalization mathematically corrects for unavoidable sample-to-sample and lane-to-lane variation by comparing the target protein to an internal loading control. The internal loading control is used as an indicator of sample protein loading, to correct for loading variation and confirm that observed changes represent actual differences between samples.</p>
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<p><strong>More Info:</strong> For more normalization related resources, see 'Further Reading' in section VII.</p>
<p> </p>
<p><strong>Using a Housekeeping Protein for Normalization</strong></p>
<p> </p>
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<p>Housekeeping proteins (HKPs) are routinely used for Western blot normalization. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. However, expression of several HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression will introduce error and may alter data analysis and interpretation.</p>
<p> </p>
<p>Before using a housekeeping protein for Western blot normalization, it is critical to validate that its expression is constant across all samples and unaffected by the specific experimental context and conditions.</p>
<p> </p>
<p>This protocol explains how to validate an HKP for use as an internal loading control, by demonstrating that HKP expression is stable in the relevant experimental samples.</p>
<p> </p>
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<p><strong>Note:</strong> This protocol is intended for use with near-infrared fluorescent Western blots.</p>
<p> </p>
<p><strong>II. Keys for Success</strong></p>
<p> </p>
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<p><strong>Saturation and linear range.</strong> Saturated bands and sample overloading frequently compromise the accuracy of QWB. Use a dilution series to verify that you are working within the linear range of detection, and signal intensity is proportional to sample loading. See the protocol: <a href="https://www.protocols.io/view/determining-the-linear-range-for-quantitative-west-g78bzrw" target="_blank">Determining the Linear Range for Quantitative Western Blot Detection </a>for more information.</p>
<p> </p>
<p><strong>Replication.</strong> Replicate samples provide information about the inherent variability of your methods, to determine if the changes you see are meaningful and significant. A minimum of three technical replicates is recommended for each sample.</p>
<p> </p>
<p><strong>Uniform sample loading.</strong> Uniform loading of total sample protein across the gel is critical for accurate QWB analysis. A protein concentration assay (BCA, Bradford, or similar assay) must be used to adjust sample concentration and load all samples as consistently as possible.</p>
<p> </p>
<p><strong>III. Required Reagents</strong></p>
<p> </p>
<p><strong>1. Treated and untreated samples</strong></p>
<div title="Page 4">Protein concentration must be determined for all samples.</div>
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<div title="Page 4"><strong>2. REVERT™ Total Protein Stain Kit (LI-COR, P/N 926-11010)</strong></div>
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<div title="Page 4">REVERT™ Total Protein Stain is used to assess sample protein loading in each lane as an internal loading control. After transfer and prior to immunodetection, the membrane is treated with this near-infrared fluorescent protein stain and imaged. Membrane staining can verify that sample protein was uniformly loaded across the gel, and assess the quality and consistency of protein transfer.</div>
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<div title="Page 4"><strong>3. Odyssey Loading Indicator, 800 nm (LI-COR, P/N 926-20002)</strong></div>
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<div title="Page 4">Odyssey Loading Indicator (OLI) is an external loading control that is added to your samples just before electrophoresis and is used to verify that a similar sample volume was loaded in each lane. Because it is an exogenous protein, it does not provide information about the amount of sample protein loaded or transferred.</div>
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<div title="Page 4"><strong>4. Electrophoresis reagents</strong></div>
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<div title="Page 4"><strong>5. Transfer reagents</strong></div>
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<div title="Page 4"><strong>6. Western blot detection reagents (near-infrared fluorescence)</strong></div>
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<div title="Page 4">Perform near-infrared Western blot detection according to the Near-Infrared Western Blot Detection Protocol</div>
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<p><a href="https://www.protocols.io/view/near-infrared-nir-western-blot-detection-pvdf-memb-g7rbzm6" target="_blank">LI-COR NIRWesternProtocol with PVDF Membrane</a></p>
<p><a href="https://www.protocols.io/view/near-infrared-nir-western-blot-detection-nitrocell-g7nbzme" target="_blank">LI-COR NIRWestern Protocol with Nitrocellulose Membrane</a></p>
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<p><strong>IV. Protocol</strong></p>
<p> </p>
<p>See 'STEPS'</p>
<p> </p>
<p><strong>V. Total Protein and HKP Quantification</strong></p>
<p> </p>
<p>See 'STEPS' (20-29)</p>
<p> </p>
<p><strong>VI. Calculations</strong></p>
<p> </p>
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<p>Prepare a spreadsheet that contains all total protein stain, HKP, and OLI quantification values using spreadsheet software.</p>
<p> </p>
<p>1. Calculate the average signal intensity for replicate samples.</p>
<p> </p>
<p>Average formula in Excel = AVERAGE(rep 1, rep 2, ...)</p>
<p> </p>
<p>Average Signal for Total Protein</p>
<p> </p>
<table>
<tbody>
<tr>
<td>Treatment</td>
<td>rep 1</td>
<td>rep 2</td>
<td>rep 3</td>
<td>Avg</td>
<td>St Dev</td>
<td>% CV</td>
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<td>0%</td>
<td>2,030,000</td>
<td>2,060,000</td>
<td>2,100,000</td>
<td>2,063,333</td>
<td>35,119</td>
<td>2%</td>
</tr>
<tr>
<td>33%</td>
<td>2,110,000</td>
<td>2,120,000</td>
<td>2,120,000</td>
<td>2,116,667</td>
<td>5,774</td>
<td>0%</td>
</tr>
<tr>
<td>66%</td>
<td>2,080,000</td>
<td>2,100,000</td>
<td>2,120,000</td>
<td>2,100,000</td>
<td>20,000</td>
<td>1%</td>
</tr>
<tr>
<td>100%</td>
<td>2,060,000</td>
<td>2,110,000</td>
<td>2,070,000</td>
<td>2,080,000</td>
<td>26,458</td>
<td>1% </td>
</tr>
</tbody>
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<p> </p>
<p>Average Signal for HKP (Tubulin)</p>
<p> </p>
<table>
<tbody>
<tr style="height: 20px;">
<td style="height: 20px;">Treatment</td>
<td style="height: 20px;">rep 1</td>
<td style="height: 20px;">rep 2</td>
<td style="height: 20px;">rep 3</td>
<td style="height: 20px;">Avg</td>
<td style="height: 20px;">St Dev</td>
<td style="height: 20px;">% CV</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">0%</td>
<td style="height: 20px;">9,190</td>
<td style="height: 20px;">9,070</td>
<td style="height: 20px;">8,780</td>
<td style="height: 20px;">9,013</td>
<td style="height: 20px;">211</td>
<td style="height: 20px;">2%</td>
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<tr style="height: 20px;">
<td style="height: 20px;">33%</td>
<td style="height: 20px;">8,210</td>
<td style="height: 20px;">7,760</td>
<td style="height: 20px;">7,340</td>
<td style="height: 20px;">7,770</td>
<td style="height: 20px;">435</td>
<td style="height: 20px;">6%</td>
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<td style="height: 14px;">66%</td>
<td style="height: 14px;">6,650</td>
<td style="height: 14px;">7,270</td>
<td style="height: 14px;">7,780</td>
<td style="height: 14px;">7,233</td>
<td style="height: 14px;">566</td>
<td style="height: 14px;">8%</td>
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<td style="height: 20px;">100%</td>
<td style="height: 20px;">7,770</td>
<td style="height: 20px;">7,590</td>
<td style="height: 20px;">7,370</td>
<td style="height: 20px;">7,577</td>
<td style="height: 20px;">200</td>
<td style="height: 20px;">3%</td>
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<p> </p>
<p>Average Signal for Odyssey Loading Indicator</p>
<p> </p>
<table>
<tbody>
<tr>
<td>Treatment</td>
<td>rep 1</td>
<td>rep 2</td>
<td>rep 3</td>
<td>Avg</td>
<td>St Dev</td>
<td>% CV</td>
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<td>0%</td>
<td>5,000</td>
<td>4,980</td>
<td>5,170</td>
<td>5,050</td>
<td>104</td>
<td>2%</td>
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<td>33%</td>
<td>5,220</td>
<td>5,320</td>
<td>5,320</td>
<td>5,287</td>
<td>58</td>
<td>1%</td>
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<td>66%</td>
<td>5,310</td>
<td>5,220</td>
<td>5,210</td>
<td>5,247</td>
<td>55</td>
<td>1%</td>
</tr>
<tr>
<td>100%</td>
<td>5,150</td>
<td>4,880</td>
<td>5,310</td>
<td>5,113</td>
<td>217</td>
<td>4%</td>
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<p> </p>
<p> </p>
<p>2. Calculate the Standard Deviation of the replicate treatment conditions.</p>
<p> </p>
<p>Standard Deviation formula in Excel = STDEV(rep 1 value, rep 2 value, ...) </p>
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<p>3. Calculate the Coefficient of Variation (CV) of the replicate treatment conditions.</p>
<p> </p>
<p>% Coefficient of Variation (CV) =<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ieec2w6.png" data-src="https://s3.amazonaws.com/pr-journal/ieec2w6.png" data-ofn="Screen Shot 2017-02-15 at 8.49.38 AM.png" /></p>
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<p> </p>
<p>4. Separately plot the relative signal intensities for each data set, HKP, total protein stain, and loading indicator (as in Figure 1).</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/iefc2w6.png" width="600" data-src="https://s3.amazonaws.com/pr-journal/iefc2w6.png" data-ofn="Screen Shot 2017-02-15 at 11.06.11 AM.png" /></p>
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<p><strong>Figure 1. HKP expression may be affected by experimental treatments.</strong> Jurkat cells were treated with etoposide to induce apoptosis. Cell lysate samples were loaded in triplicate. Relative signal intensities are shown. Intensity of tubulin bands (A) decreased by 16% across the blot, suggesting that etoposide treatment may lower the expression of this HKP. Total protein staining (B) and Odyssey Loading Indicator (C) showed consistent signals in all samples, confirming that sample protein loading and volume were uniform across the gel. This indicates that the observed changes in HKP band intensity reflect actual change in tubulin levels in the etoposide-treated cells, not variation in sample loading.</p>
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<p><strong>VII. Data Interpretation</strong> </p>
<p> </p>
<p>1. Evaluate results to determine if expression of this HKP is stable across samples. </p>
<p> </p>
<p>    a. Were samples uniformly loaded across the gel?</p>
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<ul>
<li>Consistent signals for REVERT total protein staining (as in Fig. 1B, with an approximately horizontal line) confirm that the amount of sample protein loaded was consistent and uniform.</li>
<li>Consistent signals from the Odyssey Loading Indicator (Fig. 1C) indicate that a uniform sample volume was loaded.</li>
<li>Inconsistent signals indicate a problem with sample loading, and HKP expression cannot be accurately evaluated. Repeat the Western blot experiment with uniformly loaded samples.</li>
</ul>
<p>    b. If samples were uniformly loaded, the intensity of HKP bands should reflect actual HKP expression levels in your samples.</p>
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<li>Stable HKP bands indicate that actual HKP protein levels were stable in these samples. This HKP may be suitable for Western blot normalization.</li>
</ul>
<strong>Important:</strong> The HKP must be revalidated if there are changes in experimental treatments, conditions, cell lines, etc.
<ul>
<li>Variation in HKP band intensity indicates that actual HKP expression was altered by your experimental conditions (Fig. 1A). This HKP should not be used for normalization.</li>
</ul>
<p>    c. If samples were not uniformly loaded, HKP bands may not accurately reflect actual HKP expression in these samples.</p>
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<p> </p>
<p>       HKP validation results cannot be accurately interpreted, and the Western blot experiment should be repeated with careful attention to uniform sample loading.</p>
<p> </p>
<p>2. If results indicate that HKP expression is affected by experimental conditions, this HKP should not be used for normalization. Next steps should be:</p>
<p> </p>
<div title="Page 11">    a. Repeat this process to evaluate additional HKPs, or<br />    b. Consider total protein staining of the membrane for Western blot normalization. REVERT Total Protein Stain is a robust alternative to HKPs.<br />
<ul>
<li>Total protein staining is not affected by changes in the expression of a single internal reference protein.</li>
<li>It provides an accurate readout of sample loading across a wide range, from 1 - 60 μg of total sample protein.</li>
<li>Re-validation is not required for different experimental treatments, cell lines, or other experimental parameters.</li>
</ul>
<p> </p>
<p><strong>VIII. Further Reading</strong></p>
<p> </p>
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<p>Please see the following for more information about normalization.</p>
<p> </p>
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<p><strong>Western Blot Normalization Handbook</strong></p>
<p><a href="https://www.licor.com/documents/gtk1a4mrphretj1gvmzld0f920g7h8sh" target="_blank">licor.com/handbook</a></p>
<p>The Normalization Handbook describes how to choose and validate an appropriate internal loading control for normalization.</p>
<p> </p>
<p><strong>Good Normalization Gone Bad</strong></p>
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<p><a href="https://www.licor.com/documents/r8b0v1iuo2lkvdonha1toxx778aap3tr" target="_blank">licor.com/GNGB</a></p>
<p>Good Normalization Gone Bad presents examples of normalization that have been adversely affected by common pitfalls and offers potential solutions.</p>
<p> </p>
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<p><strong>Western Blot Normalization White Paper</strong></p>
<p><a href="https://www.licor.com/documents/fwf274p9k35hwym6p2keq397p4edho5o" target="_blank">licor.com/normalizationreview</a></p>
<p>This white paper comprehensively reviews the literature of Western blot normalization.</p>
<p> </p>
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<p><strong>Determining the Linear Range for Quantitative Western Blot Detection</strong></p>
<p><a href="https://www.protocols.io/view/licor.com/LinearRange" target="_blank">licor.com/LinearRange</a></p>
<p>This protocol explains how to choose an appropriate amount of sample to load for QWB analysis.</p>
<p> </p>
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<p><strong>REVERT™ Total Protein Stain Normalization Protocol</strong></p>
<p><a href="https://www.protocols.io/view/%20licor.com/RevertNormalization" target="_blank">licor.com/RevertNormalization</a></p>
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<p>This protocol describes how to use REVERT™ Total Protein Stain for Western blot normalization and analysis.</p>
<p> </p>
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<p><strong>Pan/Phospho Analysis For Western Blot Normalization</strong></p>
<p><a href="https://www.protocols.io/view/licor.com/PanProteinNormalization" target="_blank">licor.com/PanProteinNormalization</a></p>
<p>This protocol describes how to use pan-specific antibodies as an internal loading control for normalization.</p>
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<p> </p>
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<p><strong>Housekeeping Protein Normalization Protocol</strong></p>
<p><a href="https://www.protocols.io/view/licor.com/HKP-Normalization" target="_blank">licor.com/HKP-Normalization</a></p>
<p>This protocol describes how to use a housekeeping protein for Western blot normalization and quantitative analysis.l</p>
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