TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>IRDye 800CW Maleimide is a functional derivative of infrared dye IRDye 800CW that is reactive toward free–SH (thiol, sulfhydryl) groups. Most molecules that contain free–SH groups can be labeled with maleimide dyes, including IRDye 800CW Maleimide Infrared Dye.</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>IRDye 800CW Maleimide is a functional derivative of infrared dye IRDye 800CW that is reactive toward free-SH (thiol, sulfhydryl) groups. Most molecules that contain free-SH groups can be labeled with maleimide dyes, including IRDye 800CW Maleimide Infrared Dye.</p>
<p> </p>
<p><strong>II. Labeling Reactions and Considerations</strong></p>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>Maleimide groups react with sulfhydryl groups at pH 6.5-7.5, forming a stable thioether bond. A protein, peptide, or biomolecule containing a reactive sulfhydryl group can be labeled with IRDye 800CW using the maleimide functional group of IRDye 800CW Maleimide.</p>
</div>
</div>
<div class="layoutArea">
<div class="column">
<p> </p>
<p>The following conditions provide good labeling efficiency:</p>
<p> </p>
<p><strong>Table 1.</strong> Labeling conditions for IRdye 800CW Maleimide.</p>
<p> </p>
<table>
<tbody>
<tr>
<td>Buffer:</td>
<td>Phosphate buffered Saline (PBS), pH 7.2</td>
</tr>
<tr>
<td>Temperature:</td>
<td>Ambient*</td>
</tr>
<tr>
<td>Time:</td>
<td>2 hours</td>
</tr>
<tr>
<td>Dye equivalents per free-SH</td>
<td>2-5</td>
</tr>
</tbody>
</table>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>* Ambient temperature is preferred, but 4°C may be used if the protein is not stable during incubation. If 4°C is used for the labeling reaction, an overnight incubation should be performed.</p>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>Generally, PBS works well for labeling, but other buffers with pH 6.5 to 7.5 can be used. Reactions above pH 8.0 should be avoided, since unprotonated amines can also react with maleimides. The labeling reaction is usually complete in 2 hours at room temperature, but the reaction can be carried out at 4°C for 16-18 hours. The labeled molecule should be purified by appropriate purification techniques. Dialysis, size exclusion chromatography, desalting spin columns, and HPLC all work well for purification.</p>
<p> </p>
<p>Molecules containing disulfide bonds cannot be directly labeled with a maleimide. However, the disulfide bonds can be cleaved with reducing agents such as TCEP, DTT, or 2-Mercaptoethylamine (MEM) to produce free sulfhydryl groups. After reduction, excess reducing agent should be removed prior to the labeling, since it will also react with the maleimide group.</p>
<p> </p>
<p>Additional considerations:</p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>If it is not possible to remove the reducing agent after reduction,TCEP is the preferred choice.</p>
</li>
<li>
<p>Contrary to some claims in the literature, we have observed that TCEP will react with the maleimide group of the dye during labeling reaction. The ratio of TCEP and maleimide dye to the protein or peptide must be optimized for efficient reduction and labeling.</p>
</li>
<li>
<p>The overall concentrations of the reactants affect the rate of the reaction.</p>
</li>
</ul>
<p> </p>
<p><strong>III. Examples</strong></p>
<p> </p>
<p><strong>Labeling of Affibody® Molecules with IRDye 800CW Maleimide</strong></p>
<p>Affibody molecules are small proteins with unique binding sites capable of binding to different target proteins (<a href="http://www.affibody.com" target="_blank" rel="noopener noreferrer">www.affibody.com</a>). Commercial Affibody molecules are engineered with a single C-terminal cysteine residue that can be coupled to any fluorescent dye. The Affibody molecules are partially dimerized due to S-S bridges formed by the C-terminal cysteine and must be reduced prior to labeling with IRDye 800CW Maleimide.</p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column"> </div>
<div class="column">See 'STEPS'</div>
<div class="column"> </div>
<div class="column"><strong>Labeling of Small Molecules with IRDye 800CW Maleimide</strong></div>
<div class="column">
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Glutathione is a small peptide which is available in the reduced form. LI-COR has used glutathione as a model compound to optimize the labeling of small molecules containing free thiols with IRDye 800CW Maleimide. The following procedure should serve as a general guideline for labeling small molecules containing a free-SH group.</p>
<p> </p>
<p>See 'STEPS'</p>
<p> </p>
<p><strong>Labeling of Antibodies with IRDye 800CW Maleimide</strong></p>
<div class="page" title="Page 3">
<div class="layoutArea">
<div class="column">
<p>Antibodies can be labeled with IRDye 800CW Maleimide by selective reduction of disulfide bonds in the hinge region using 2-Mercaptoethylamine (MEM). 2-Mercaptoethylamine is a mild reducing agent which selectively reduces the two disulfide bonds in the hinge region of IgG, thereby producing two heavy chain-light chain molecules, each containing one antigen binding site.</p>
<p> </p>
<p>See 'STEPS'</p>
<p> </p>
<p><em><strong>For futher questions, please send a detailed inquiry to <a href="mailto:biohelp@licor.com" target="_blank" rel="noopener noreferrer">biohelp@licor.com</a></strong></em></p>
<p> </p>
<p><strong>References</strong></p>
<div class="page" title="Page 4">
<div class="layoutArea">
<div class="column">
<ol>
<li>
<p>E. B. Getz, M. Xiao,T. Chakrabarty, R. Cooke, P. R. Selvin. 1999. A comparison between the sulfhydryl reductants tris (2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273: 73-80</p>
</li>
<li>
<p>J. C. Han et al. 1994. A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal. Biochem. 220: 5-10</p>
</li>
<li>
<p>T. L. Kirley. 1989. Reduction and fluorescent labeling of cysteine containing proteins for subsequent structural analysis. Anal. Biochem. 180: 231-236</p>
</li>
<li>
<p>M.E. Levison et al. 1969. Reduction of biological samples by water-soluble phosphines: Gammaglobulin. Experiantia. 25: 126-127</p>
</li>
<li>
<p>J. C. Mery et al. 1993. Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical application of peptide,resins and mercaptoamide peptides. Int. J. Peptide Protein Res. 42: 44-52</p>
</li>
<li>
<p>D. E. Shafer, J. K. Inman, A. Lees. 2000. Reaction ofTris(2-carboxyethyl)phosphine (TCEP) with Maleimide and alphahaloacylgroups: anomalous elution ofTCEP by gel filtration. Anal. Biochem. 282: 161-164</p>
</li>
<li>
<p>R. Scherer. 2008. LI-COR IRDye® 800CW Maleimide conjugation report. Int. Materials Science &amp; Engineering, Vanderbilt University.</p>
</li>
<li>
<p>K.Tyagarajan, E. Pretzer, J. E.Wiktorowicz. 2003. Thiol-reactive dyes for fluorescence labeling of proteomic samples. Electrophoresis. 24: 2348-2358</p>
</li>
<li>
<p>P. Blauenstein, et al. 1995. Experience with the iodine-123 and technitium-99 m labeled anti-granulocyte antibody Mab47: a comparison of labeling methods. Eur. J. nuclear Med. 22:690-698.</p>
</li>
<li>10.G. Hermanson. 1996. BioconjugateTechniques.</li>
</ol>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Affibody® Molecules with IRDye 800CW Maleimide
?. 
SECTION: IRDye 800CW Maleimide Labeling
?. 
SECTION: Labeling of Small Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Small Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Small Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Small Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Small Molecules with IRDye 800CW Maleimide
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Reduction
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Reduction
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Reduction
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Reduction
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Labeling
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Labeling
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Labeling
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Labeling
?. 
SECTION: Labeling of Antibodies with IRDye 800CW Maleimide/Labeling
?.