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[DESCRIPTION]
<p>IRDye NHS esters react with unblocked amines in peptides. NHS ester reactions usually proceed quickly and cleanly in either organic or aqueous solvents. While NHS esters continue to be widely used tools for biomolecule modification, their application to large peptides may be complicated by factors such as steric hindrance, amine inactivation by salt-bridging, and multiple labeling sites.</p>
<p> </p>
<p>Although a properly designed peptide can be labeled site-specifically by NHS ester chemistry, complex designs may incur cost-prohibitive materials and labor.To address the limitations of NHS ester chemistry, orthogonal technologies such as IRDye Maleimides and Click Chemistry Reagents have been developed to label non-amine functional groups. Orthogonal chemistry can also facilitate dual- or multi-dye labeling. As with NHS ester chemistry, optimal non-amine labeling is usually achieved by aqueous solution-phase reactions.</p>
<p> </p>
<p>Developed for: </p>
<p> </p>
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<p>Odyssey® Classic, Odyssey CLx, Odyssey Sa, and Aerius Imaging Systems</p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
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<p>Peptides labeled with fluorescent dyes are important as probes for <em>in vivo</em> imaging and as substrates for enzyme activity assays. Near-infrared (NIR) fluorophores such as IRDye 800CW (excitation 780 nm; emission 794 nm) can offer improved sensitivity because of low NIR autofluorescence from tissues, cells, biological materials, or drug compounds. Fluorophores can also be combined with appropriate quencher dyes to create fluorogenic peptide probes, such as those used for protease activity detection.<sup>1-4</sup> IRDye QC-1 can efficiently quench a wide range of fluorophores spanning the visible and NIR spectra (~500 - 800 nm) in a fluorescence reso- nance energy transfer (FRET) system.Together, IRDye 800CW and IRDye QC-1 comprise an optimal fluorophore-quencher pair for incorporation into NIR fluorogenic peptides (Figure 1).</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/heuc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/heuc2w6.png" data-ofn="Screen Shot 2016-12-21 at 10.51.42 AM.png" /></p>
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<p>IRDye fluorophores and quenchers are amenable to a wide variety of conjugation reactions that furnish stable, covalently-labeled peptides. Furthermore, site-specific dye labeling can be achieved by employing orthogonally reactive groups in peptide design. Because IRDye labels have been optimized for detection in aqueous biological environments, conjugation reactions are best performed in solution-phase on fully deprotected peptides.</p>
<p> </p>
<p><strong>II. Peptide Labeling Options</strong></p>
<p> </p>
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<p><strong>IRDye NHS esters react with unblocked amines in peptides.</strong> NHS ester reactions usually proceed quickly and cleanly in either organic or aqueous solvents. While NHS esters continue to be widely used tools for biomolecule modification, their application to large peptides may be complicated by factors such as steric hindrance, amine inactivation by salt-bridging, and multiple labeling sites.</p>
<p> </p>
<p>Although a properly designed peptide can be labeled site-specifically by NHS ester chemistry, complex designs may incur cost-prohibitive materials and labor.To address the limitations of NHS ester chemistry, orthogonal technologies such as IRDye Maleimides and Click Chemistry Reagents have been developed to label non-amine functional groups. Orthogonal chemistry can also facilitate dual- or multi-dye labeling. As with NHS ester chemistry, optimal non-amine labeling is usually achieved by aqueous solution-phase reactions.Table 1 summarizes general strategies for labeling peptides with IRDye reagents.</p>
<p> </p>
<p><strong>Table 1.</strong> IRDye Reagents for Labeling Peptides</p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p><strong>Desired Peptide</strong></p>
<p><strong>Labeling Site</strong></p>
</td>
<td>
<p><strong>Appropriate</strong></p>
<p><strong>IRDye Reagent</strong></p>
</td>
<td><strong>Notes</strong></td>
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<td>
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<p>Unblocked N-terminus or</p>
<p>Side-Chain Amine (e.g., lysine)</p>
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</td>
<td>
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<p>NHS Ester</p>
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</td>
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<p>Perform in mildly basic aqueous solutions; peptides</p>
<p>containing &gt;1 amine may yield product mixtures</p>
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</td>
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<td>
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<p>Sulfhydryl (e.g., cysteine)</p>
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</div>
</td>
<td>
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<p>Maleimide</p>
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</td>
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<p>Perform in mildly acidic aqueous solutions</p>
<p>for selective labeling in the presence of amines</p>
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</div>
</div>
</td>
</tr>
<tr>
<td>
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<p>Alkyne</p>
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</td>
<td>
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<p>Azide</p>
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</td>
<td>
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<p>Copper-catalyzed Click Chemistry for selective</p>
<p>labeling in the presence of amines</p>
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</td>
</tr>
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<p> </p>
<p>Azide</p>
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 </td>
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<p>Alkyne</p>
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</td>
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<p>Copper-catalyzed Click Chemistry for selective</p>
<p>labeling in the presence of amines</p>
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</td>
</tr>
<tr>
<td> DBCO</td>
<td>Copper-free Click Chemistry for selective<br />
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<p>labeling in the presence of amines</p>
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</td>
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<td>
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<p>DBCO</p>
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</td>
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<p>Azide</p>
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</div>
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</td>
<td>
<p> Copper-free Click Chemistry for selective labeling</p>
<p>in the presence of amines</p>
</td>
</tr>
</tbody>
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<p><strong>IRDye Maleimides are designed to react selectively with sulfhydryl groups in the presence of amines.</strong> Optimal selectivity is usually obtained with labeling reactions performed in mildly acidic (pH 6.5) aqueous solvents; at acidic pH, amines are rendered inert by protonation. Sulfhydryl groups can be installed in peptides with cysteine or commercially-available thiollinkers.</p>
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<p><em>NOTE: Peptides containing sulfhydryls can spontaneously form inactive dimers.Therefore, it may be necessary to pre-treat the peptide with a reducing agent such as tris(2-carboxyethyl) phosphine (TCEP) to reveal active monomers.</em></p>
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<p><strong>IRDye Click Chemistry reagents are mostly inert to naturally-occurring functional groups such as amines and hydroxyls.</strong> Click Chemistry utilizes high-fidelity pairs of functional groups that can react either in the presence<sup>5</sup> or absence<sup>6</sup> of a copper catalyst. Click Chemistry functional groups can also be readily installed in peptides with commercially-available reagents.</p>
<p> </p>
<p><strong>III. Labeling Reaction Techniques</strong></p>
<p><strong>a. Aqueous Solution-Phase Labeling</strong></p>
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<p>For all labeling reactions, it is critical to omit components that can interfere with the coupling of the peptide and the dye. Peptides should be dissolved in aqueous buffers that do not contain extraneous nucleophiles such as Tris, sodium azide, DTT, BME, etc.To maximize the labeling efficiency, the peptide should be dissolved at the highest practical concentration that still maintains a homogeneous reaction mixture.The aqueous reaction mixture can be directly purified by reverse-phase HPLC (see Section III.C. Purification).Table 2 provides general parameters for aqueous solution-phase labeling of peptides with IRDye reagents.</p>
<p> </p>
<p><strong>Table 2.</strong> General parameters for labeling peptides in aqueous solutions</p>
<p> </p>
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<p><strong>IRDye </strong></p>
<p><strong>Reagent</strong></p>
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<p><strong>Dye Equivalents </strong></p>
<p><strong>per Labeling Site</strong></p>
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<p><strong>Reaction </strong></p>
<p><strong>Buffer (pH)</strong></p>
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<p><strong>Reaction </strong></p>
<p><strong>Temperature</strong></p>
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</td>
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<p><strong>Reaction </strong></p>
<p><strong>Time (hours)</strong></p>
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</td>
</tr>
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<p>NHS Ester</p>
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</td>
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<p>Phosphate</p>
<p>(8.0–8.5)</p>
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</td>
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<p>Ambient</p>
<p>to 37 °C</p>
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</div>
</td>
<td style="text-align: center; width: 100px; height: 32px;">2-3</td>
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<p>Maleimide</p>
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</td>
<td style="text-align: center; width: 126px; height: 32px;">1-2</td>
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<p>Phosphate</p>
<p>(6.5–7.5)</p>
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<p>Ambient</p>
<p>to 37 °C</p>
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</div>
</td>
<td style="text-align: center; width: 100px; height: 32px;">2-3</td>
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<p>Alkyne†</p>
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</td>
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<p>Variable</p>
<p>(6.5 to 8.5)</p>
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</td>
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<p>Ambient</p>
<p>to 37 °C</p>
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</div>
</div>
</td>
<td style="text-align: center; width: 100px; height: 32px;">1-2</td>
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<p>Azide†,*</p>
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</td>
<td style="text-align: center; width: 126px; height: 32px;">1</td>
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<p>Variable</p>
<p>(6.5 to 8.5)</p>
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</div>
</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 100px; height: 32px;">1-2</td>
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<p>DBCO*</p>
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</div>
</div>
</td>
<td style="text-align: center; width: 126px; height: 32px;">1</td>
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<p>Variable</p>
<p>(6.5 to 8.5)</p>
</div>
</div>
</div>
</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 100px; height: 32px;">1-2</td>
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<p>* Copper-catalyzed Click Chemistry<sup>5 </sup></p>
<p>† Copper-free Click Chemistry<sup>6</sup></p>
<p> </p>
<p><strong>b. Organic Solution-Phase Labeling</strong></p>
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<p>Certain peptides (e.g., comprised mainly of hydrophobic amino acids) may not be amenable to aqueous solution-phase labeling. In these atypical cases, IRDye reagents can be used for organic solution-phase labeling; however, this reaction may be slower than its aqueous counterpart. Again, peptides should be dissolved in solvents devoid of extraneous nucleophiles such asTris, sodium azide, DTT, BME, etc.The optimal reaction solvent is anhydrous dimethyl sulfoxide (DMSO). When labeling with IRDye NHS esters and Maleimides, including a tertiary amine base such as N,N-diisopropylethylamine (DIPEA) is necessary to promote the reaction. After the reaction has completed, the crude IRDye labeled peptide can be precipitated from the DMSO by dropwise addition of anhydrous diethyl ether. IRDye labels impart hydrophilicity and can facilitate the purification of hydrophobic peptides by reverse-phase HPLC (see Section III.c. Purification).Table 3 provides general parameters for organic solution-phase labeling of peptides with IRDye reagents.</p>
<p> </p>
<p><strong>Table 3.</strong> General parameters for labeling peptides in organic solutions</p>
<p> </p>
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<p><strong>IRDye </strong></p>
<p><strong>Reagent</strong></p>
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<p><strong>Dye Equivalents </strong></p>
<p><strong>per Labeling Site</strong></p>
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</td>
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<p><strong>Base Equivalents</strong></p>
<p><strong>per Labeling Site</strong></p>
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</td>
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<p><strong>Reaction </strong></p>
<p><strong>Temperature</strong></p>
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</div>
</td>
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<p><strong>Reaction </strong></p>
<p><strong>Time (hours)</strong></p>
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</div>
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<p>NHS Ester</p>
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</div>
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</td>
<td style="text-align: center; width: 129px;">1</td>
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<p>2 - 4</p>
</div>
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</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 137px;">2 - 12</td>
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<p>Maleimide</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 129px;">1</td>
<td style="text-align: center; width: 143px;">
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<p>2 - 4</p>
</div>
</div>
</div>
</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 137px;">2 - 12</td>
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<p>Alkyne†</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 129px;">1</td>
<td style="text-align: center; width: 143px;">
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<p> </p>
None</div>
</div>
</div>
</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 137px;">1 - 4</td>
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<p>Azide†,*</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 129px;">1</td>
<td style="text-align: center; width: 143px;">
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<p> </p>
None</div>
</div>
</div>
</td>
<td style="text-align: center; width: 93px;">
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 137px;">1 - 4</td>
</tr>
<tr>
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<p>DBCO*</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 129px;">1</td>
<td style="text-align: center; width: 143px;">
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<p> </p>
None</div>
</div>
</div>
</td>
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<p>Ambient</p>
<p>to 37 °C</p>
</div>
</div>
</div>
</td>
<td style="text-align: center; width: 137px;">1 - 4</td>
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<p>* Copper-catalyzed Click Chemistry<sup>5 </sup></p>
<p>† Copper-free Click Chemistry<sup>6</sup></p>
<p> </p>
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<p><strong>For questions, please send a detailed inquiry to biohelp@licor.com</strong></p>
<p> </p>
<p><strong>c. Purification</strong></p>
<p> </p>
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<p>Reverse-phase HPLC purification delivers IRDye dye-labeled peptides with the highest purity. Zorbax C18 HPLC columns (Agilent Technologies) work well, and are available in different sizes for analytical and preparatory scale.The recommended mobile phase is a gradient mixture of acetonitrile and water buffered with triethylammonium acetate (TEAA, 50 mM, pH 6.0).The TEAA provides ion-pairing for better retention behavior of the hydrophilic IRDye dye-labeled peptides on the column. As a consequence, however, the predominant counter-ion will be triethylammonium, which may interfere with downstream biological experiments. Prior to lyophilization, the purified peptide should be ion-exchanged by eluting through an appropriate resin (e.g., Amberlite™) or by dialysis. For flexibility in development and/or troubleshooting, the HPLC system should be equipped with a diode array detector (DAAD) and be able to monitor at the absorption maxima of all dyes used.</p>
<p><strong>For questions, please send a detailed inquiry to biohelp@licor.com</strong></p>
<p> </p>
<p><strong>d. Quantification</strong></p>
<p> </p>
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<p>UV-Vis absorbance spectroscopy is the best method for quantifying the amount of purified IRDye labeled peptide.To determine the concentration of a stock solution containing a singly-labeled peptide, dilute an aliquot of the stock solution in 1X PBS, measure the absorbance spectra at the dye-specific maximum (baseline subtraction from 950 to 1000 nm), and use the following equation:</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hevc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hevc2w6.png" data-ofn="Screen Shot 2016-12-21 at 11.08.49 AM.png" /></p>
<p> </p>
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<div class="column">
<p>In which:</p>
<ul>
<li>
<p>A<sub>dye</sub> is the measured absorbance at the dye-specific maximum (See Table 4)</p>
</li>
<li>ε<sub>dye</sub> is the extinction coefficient for the dye in 1X PBS (See Table 4)</li>
<li>
<p>Dilution Factor is the fold dilution of the IRDye peptide in 1X PBS</p>
</li>
<li>
<p>MW<sub>IRDye peptide</sub> is the molecular weight of the IRDye peptide</p>
</li>
</ul>
<p> </p>
<p><strong>Table 4.</strong> Parameters for UV-Vis quantification of IRDye labeled peptides in 1X PBS</p>
<p> </p>
<table>
<tbody>
<tr>
<td><strong>Label</strong></td>
<td>
<p><strong>Dye Maximum (nm) for </strong></p>
<p><strong>Measuring Absorbance</strong></p>
</td>
<td>
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<div class="layoutArea">
<div class="column">
<p><strong>e <sub>dye</sub></strong></p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 800CW</p>
</div>
</div>
</div>
</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>774</p>
</div>
</div>
</div>
</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>240,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 800RS</p>
</div>
</div>
</div>
</td>
<td>767</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>200,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 750</p>
</div>
</div>
</div>
</td>
<td>756</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>260,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 700DX</p>
</div>
</div>
</div>
</td>
<td>689</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>165.000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 680RD</p>
</div>
</div>
</div>
</td>
<td>672</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>165,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 680LT</p>
</div>
</div>
</div>
</td>
<td>676</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>250,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye 650</p>
</div>
</div>
</div>
</td>
<td>648</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>230,000</p>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>IRDye QC-1</p>
</div>
</div>
</div>
</td>
<td>737</td>
<td>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>96,000</p>
</div>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p><strong>For a pure stock solution of a FRET-quenched peptide labeled with exactly one IRDye 800CW and one IRDye QC-1:</strong> Determine the concentration of a stock solution by diluting an aliquot of the stock solution in methanol, measuring the absorbance spectra at 778 nm and 850 nm (baseline subtraction from 950 to 1000 nm), and using the following equation:</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hewc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hewc2w6.png" data-ofn="Screen Shot 2016-12-21 at 11.13.38 AM.png" /></p>
<p> </p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<p>In which:</p>
<ul>
<li>
<p>A<sub>778</sub> is the measured absorbance at the maximum for IRDye 800CW in methanol</p>
</li>
<li>
<p>300,000 is the extinction coefficient for IRDye 800CW in methanol</p>
</li>
<li>
<p>1.265 is the correction factor for the ratio of A<sub>778/A850</sub> for IRDye QC-1 in methanol</p>
</li>
<li>
<p>A<sub>850</sub> is the measured absorbance at the maximum for IRDye QC-1 in methanol</p>
</li>
<li>
<p>Dilution Factor is the fold dilution of the IRDye 800CW QC1-peptide in methanol</p>
</li>
<li>
<p>MWI<sub>RDye 800CW QC1 peptide</sub> is the molecular weight of the IRDye 800CW QC1 peptide</p>
<p><strong>For questions, please send a detailed inquiry to biohelp@licor.com</strong></p>
</li>
</ul>
<p> </p>
<p><strong>IV. Examples</strong></p>
<div class="page" title="Page 6">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p><strong>For an example of aqueous solution-phase labeling with IRDye 800CW NHS ester, see:</strong> Davies-Venn CA, Angermiller B, Wilganowski N, Ghosh P, Harvey BR, Wu G, Kwon S, Aldrich MB, Sevick-Muraca EM. Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging. Mol. Imaging Biol. 14(2012), 301 – 314.</p>
</li>
<li>
<p><strong>For an example of organic solution-phase labeling with IRDye 800CW NHS ester, see:</strong> Chen Y, Dhara S, Banerjee SR, Byun Y, Pullambhatla M, Mease RC, Pomper M. A low molecular weight PSMA-based fluorescent imaging agent for cancer. Biochem. Biophys. Res. Commun. 390(2009), 624–629.</p>
</li>
<li>
<p><strong>For an example of aqueous solution-phase labeling with IRDye 800CW Maleimide, see:</strong> YeY, Zhu L, MaY, Niu G, Chen X. Synthesis and evaluation of new iRGD peptide analogs for tumor optical imaging. Bioorg. Med. Chem. Lett. 21(2011), 1146–1150. </p>
</li>
<li><strong>For examples of dual-labeling with IRDye® QC-1 NHS ester and various fluorophores, see:</strong> Sun X, Zhang A, Baker B, Sun L, Howard A, Buswell J, Maurel D, Masharina A,Johnsson K, Noren CJ, Xu M-Q, Corrêa IR. Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging. ChemBioChem 12 (2011), 2217–2226.</li>
</ul>
<p> </p>
<p><strong>V. Additional References</strong></p>
<div class="page" title="Page 7">
<div class="layoutArea">
<div class="column">
<ol>
<li>
<p>Matayoshi ED, Wang GT, Krafft GA, Erickson J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247(1990), 954-958.</p>
</li>
<li>
<p>Bullok KE, Maxwell D, Kesarwala AH, Gammon S, Prior JL, Snow M, Stanley S, Piwnica-Worms D. Biochemical and in vivo characterization of a small, membrane- permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. Biochem. 46 (2007), 4055-4065.</p>
</li>
<li>
<p>Zheng G, Chen J, Stefflova K, Jarvi M, Li H, Wilson BC. Photodynamic molecular beacon as an activatable photosensitizer based on protease controlled singlet oxygen quenching and activation. Proc. Natl. Acad. Sci. USA 104 (2007), 8989-8994.</p>
</li>
<li>
<p>Blum G, Mullins SR, Keren K, Fonovic M, Jedeszko C, Rice MJ, Sloane BF, Bogyo M. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nature Chem. Biol. 1(2005), 203-209.</p>
</li>
<li>
<p>Best MD. Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules. Biochemistry 48(2009), 6571-6584.</p>
</li>
<li>
<p>Simon M, Zangemeister-Wittke U, Plückthun A. Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click andThiol Chemistries. Bioconjugate Chem. 23(2012), 279-286.</p>
</li>
</ol>
<p> </p>
<p><strong>VI. Associated Products</strong></p>
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<div class="column">
<ul>
<li><a href="http://www.licor.com/bio/products/reagents/nhs_esters/nhs_esters.jsp" target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/products/reagents/nhs_esters/nhs_esters.jsp</a></li>
<li><a href="http://www.licor.com/bio/products/reagents/maleimides/maleimides.jsp" target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/products/reagents/maleimides/maleimides.jsp</a></li>
<li><a href="http://www.licor.com/bio/products/reagents/click_chemistry/clickChemistryOverview.jsp " target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/products/reagents/click_chemistry/clickChemistryOverview.jsp </a></li>
<li><a href="http://www.licor.com/bio/products/reagents/fret_based_assays/fret_based_assays.jsp" target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/products/reagents/fret_based_assays/fret_based_assays.jsp</a></li>
<li><a href="http://www.licor.com/bio/services/custom_labeling.jsp" target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/services/custom_labeling.jsp</a></li>
<li><a href="http://www.licor.com/bio/network/#crb" target="_blank" rel="noopener noreferrer">http://www.licor.com/bio/network/#crb</a></li>
</ul>
</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>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
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[STEPS]
SECTION: Aqueous Solution-Phase Labeling
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SECTION: Aqueous Solution-Phase Labeling
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SECTION: Organic Solution-Phase Labeling
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SECTION: Organic Solution-Phase Labeling
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SECTION: Organic Solution-Phase Labeling
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SECTION: Purification
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SECTION: Quantification
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