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[DESCRIPTION]
<p>This protocol is a guide to using IRDye Subclass Specific antibodies for In-Cell Western™ Assay (ICW) applications. For more detailed descriptions of ICW techniques, refer to Western Blot Analysis and In-Cell Western Kits I and II on the LI-COR Biosciences website (<a href="http://www.licor.com" target="_blank">www.licor.com</a>). </p>
<p> </p>
<p>Developed for: Odyssey® Family of Imagers</p>

[GUIDELINES]
<p><strong>I. Introduction</strong></p>
<p> </p>
<p>IRDye Goat anti-Mouse IgG1, Goat anti-Mouse IgG2a and Goat anti-Mouse IgG2b, allow for two-color detection using primary antibodies derived from the same species (mouse). IRDye Subclass Specific antibodies react with the heavy (gamma) chain only of the primary antibody. In mice, there are five unique subclasses of IgG; IgG<sub>1</sub>, IgG<sub>2a</sub>, IgG2b, IgG<sub>2c</sub> and IgG<sub>3</sub>. Each subclass is based on small differences in amino acid sequences in the constant region of the heavy chains so antibodies directed against a particular subclass will not recognize antibodies directed against other subclasses. For example, IRDye goat anti-mouse IgG<sub>1</sub> recognizes mouse gamma 1; it will not recognize mouse gamma 2a, 2b, 2c or gamma 3. All other LI-COR IRDye secondary antibodies are whole IgG (H + L) and react with the heavy (gamma) and light (kappa or lambda) chains of the primary antibody. Figure I demonstrates the differences in detection between the IRDye antibodies.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hd9c2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hd9c2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.34.40 AM.png" /></p>
<p> </p>
<p>Antibody Subclasses may also be designated by their light chains. There are two types of light chains, kappa (κ) or lambda (λ). In mice, 95% of light chains are kappa and 5% are lambda. These subclasses still contain the heavy (gamma) portion of the antibody so IRDye Subclass Specific antibodies still recognize them. If the subclass of the primary antibody is unknown, LI-COR® whole IgG secondary antibodies may be used since they recognize most mouse IgG subclasses.</p>
<p> </p>
<p><strong>II. Suggested Materials</strong></p>
<p> </p>
<p>This section is intended as a guideline; other materials may be substituted, if desired.</p>
<ul>
<li>Proteins transferred to a nitrocellulose or PVDF membrane (for Western blot only)</li>
<li>Cells that have been fixed and permeabilized on a 96 well plate (for ICW only)</li>
<li>Odyssey® Blocking Buffer</li>
<li>10X PBS</li>
<li>20% Tween® 20</li>
<li>SDS (if using PVDF membrane)</li>
<li>Suggested mouse primary antibodies for normalization:</li>
</ul>
<p>           – Beta-Actin Mouse mAb IgG<sub>2b</sub> (LI-COR P/N 926-42212)</p>
<p>           – Alpha-Tubulin Mouse mAb IgG<sub>1</sub> (LI-COR P/N 926-42213)</p>
<ul>
<li>One or two of the following IRDye secondary antibodies</li>
</ul>
<table>
<tbody>
<tr>
<td><strong>Description</strong></td>
<td><strong>LI-COR Part Number</strong></td>
</tr>
<tr>
<td>IRDye 800CW Goat anti-Mouse IgG<sub>1</sub> Specific</td>
<td>926-32350</td>
</tr>
<tr>
<td>IRDye 800CW Goat anti-Mouse IgG<sub>2a</sub> Specific</td>
<td>926-32351</td>
</tr>
<tr>
<td>IRDye 800CW Goat anti-Mouse IgG<sub>2b</sub> Specific</td>
<td>926-32352</td>
</tr>
<tr>
<td>IRDye 680LT Goat anti-Mouse IgG<sub>1</sub> Specific</td>
<td>926-68050</td>
</tr>
<tr>
<td>IRDye 680LT Goat anti-Mouse IgG<sub>2a</sub> Specific</td>
<td>926-68051</td>
</tr>
<tr>
<td>IRDye 680LT Goat anti-Mouse IgG<sub>2b</sub> Specific</td>
<td> 926-68052</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>III. Western Blot Detection</strong></p>
<p> </p>
<p>IRDye Subclass Specific antibodies are easily incorporated into the detection step of any Western blot protocol. The sample protocol provided <a href="https://www.protocols.io/view/western-blot-detection-gupbwvn" target="_blank">here</a>, optimized for LI-COR reagents, is recommended. After protein transfer to the membrane is complete, perform the <a href="https://www.protocols.io/view/western-blot-detection-gupbwvn" target="_blank">Western Blot Detection protocol</a> for one- or two-color detection.</p>
<p> </p>
<p><strong>IV. Two-Color Western Blot Considerations</strong></p>
<p> </p>
<p>Two different antigens can be detected simultaneously on the same blot using IRDye Subclass Specific OR IRDye whole IgG antibodies that are visualized in different fluorescence channels (700 and 800 nm). Two-color detection requires careful selection of primary and secondary antibodies. The following guidelines will help with the design of two-color experiments:</p>
<ul>
<li>If the two primary antibodies are monoclonals (mouse) and are IgG<sub>1</sub>, IgG<sub>2a</sub> or IgG<sub>2b</sub>, IRDye Subclass Specific secondary antibodies must be used. The same subclasses cannot be combined in a two-color Western blot (for example, two IgG<sub>1</sub> primary antibodies).</li>
<li>If the two primary antibodies are derived from different host species (for example, primary antibodies from mouse and chicken), IRDye whole IgG secondary antibodies derived from the same host and labeled with different IRDye fluorophores must be used (for example, IRDye 800CW Donkey anti-mouse and IRDye 680LT Donkey anti-chicken).</li>
<li>Before combining primary antibodies in a two-color experiment, always perform preliminary blots with each primary antibody alone to determine the expected banding pattern and possible non-specific background bands.</li>
</ul>
<p> </p>
<p>Figures II (A-C) and III (A-C) demonstrate two-color Western blot detection using (A) IRDye Subclass Specific antibodies and (B) IRDye whole IgG antibodies, respectively. IRDye Subclass Specific secondary antibodies should NOT be used in combination with IRDye whole anti-mouse IgG secondary antibodies for two-color detection. IRDye whole anti-mouse IgG secondary antibodies and IRDye Subclass Specific secondary antibodies both recognize the gamma chain of the primary antibody, causing detection in both channels (C). IRDye Subclass Specific antibodies can be used in combination with IRDye whole goat anti-rabbit secondary antibodies.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/heac2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/heac2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.45.40 AM.png" /></p>
<p> </p>
<p>Two-color Western blot detection can be achieved by multiplexing LI-COR® mouse primary antibodies and IRDye Subclass Specific antibodies. Figure IV demonstrates two-color detection utilizing the LI-COR mouse primaries and IRDye Subclass Specific secondaries.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hebc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hebc2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.46.19 AM.png" /></p>
<p> </p>
<p><strong>V. ICW Detection and Considerations</strong></p>
<p><strong> </strong></p>
<p>In-Cell Western™ assays commonly use primary and secondary antibodies for normalization in the 700 channel. For example, if phospho-ERK is the target of interest, an antibody against total ERK (or against a housekeeping protein) can be used to normalize for variations in cell number. IRDye Subclass Specific antibodies can be incorporated into the detection step of any ICW protocol. A recommended protocol is provided in 'STEPS'. After cells have been fixed and permeabilized, perform the 'STEPS'.</p>
<p> </p>
<p>The same considerations for two-color Western blot detection apply to two-color In-Cell Western™ detection with the following addition:</p>
<ul>
<li>Choose primary antibodies that have been recommended for other immunofluorescence techniques such as IF-IC and IHC.  </li>
<li>Establish the specificity of the primary antibody by screening lysates through Western blotting and detection on an Odyssey Imaging system. To achieve the most consistent results, use the same blocking buffer for validation experiments and In-Cell Western assays. If significant non-specific binding is detected on a Western blot, choose alternative primary antibodies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results.</li>
</ul>
<p>Figures V and VI demonstrate In-Cell Western Assay data generated using MitoSciences’ MitoBiogenesis™ In-Cell ELISA Kit (IR). The kit utilizes IRDye Subclass Specific antibodies for detection.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hecc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hecc2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.55.37 AM.png" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hedc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hedc2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.56.09 AM.png" /></p>
<p> </p>
<p>Figure VII compares IRDye whole IgG vs. IRDye Subclass Specific antibody detection by ICW. Extracellular-signal related kinase (ERK) phosphorylation was measured following the LI-COR protocol entitled, In-Cell Western Assay For Assessing Response of A431 Cells to Stimulation with Epidermal Growth Factor. This document can be found on the LI-COR website (Doc# 988-11453). All primary antibodies were qualified by Western blot prior to ICW (data not shown).</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/heec2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/heec2w6.png" data-ofn="Screen Shot 2016-12-20 at 11.57.03 AM.png" /></p>

[STEPS]
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