Principles of Bioorthogonal Labeling and Click Chemistry

The amine- and thiol-reactive labeling chemistries described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 and Thiol-Reactive Probes—Chapter 2 are generally used in one of two ways: (1) labeling of purified proteins or other biopolymers yielding conjugates that are subsequently applied to cell or tissue specimens, or (2) nonselective in situ labeling of total cellular thiol or amine content. In situ labeling of specific molecular populations—such as proteins and nucleic acids that have been newly synthesized in some experimental time window of interest—is not feasible due to the ubiquitous distribution of amines and thiols in cells, as well as in the media in which they are maintained. Click-iT labeling technology overcomes this obstacle by employing bioorthogonal reactive chemistry, in which the reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms.ref In addition to reaction selectivity, in situ labeling methods should allow reactivity under mild conditions and in predominantly aqueous solvent conditions.

Although several known chemistries fulfill the requirements described above,ref Click-iT labeling technology is founded upon one of the most successful and versatile bioorthogonal labeling reactions currently available—the copper-catalyzed azide–alkyne cyloaddition ref (Figure 3.1.1). Application of this reaction to in situ labeling of cells is a two-step process. First, one reaction partner—either an azide or alkyne linked to a "building block" such as a nucleotide, nucleoside, amino acid, monosaccharide or fatty acid—is biosynthetically incorporated. Subsequently, the other reaction partner—the complementary alkyne or azide linked to a fluorescent dye, biotin or other detection reagent—is "clicked" into place in the presence of catalytic copper (I). One reaction partner must be an azide derivative and the other an alkyne derivative, but either functional moiety can serve as the biosynthetically incorporated molecule or the detection molecule ref (e.g., L-azidohomoalanine (AHA) + Alexa Fluor 488 alkyne is the inverse of the reaction scheme shown in Figure 3.1.1B).

An important aspect of the azide and alkyne reaction partners is their small size (Figure 3.1.2). Expression tags such as Green Fluorescent Protein (GFP) provide the ultimate in labeling specificity because their linkage to proteins of interest is genetically prescribed. Once the GFP transgene has been inserted into a cell (BacMam Gene Delivery and Expression Technology—Note 11.1), in situ labeling is obtained without any outside intervention using the cellular transcription and translation machinery. However, the finite size of GFP (~27,000 daltons) sometimes causes functional perturbations and has spurred the development of alternative, smaller-sized expression tags such as the TC-FlAsH tetracysteine tag and biarsenical ligand system (T34561, T34562, T34563; Thiol-Reactive Probes Excited with Visible Light—Section 2.2). Furthermore, nucleic acids, lipids, glycans and post-translational protein modifications can only be detected indirectly by genetically encoded protein reporters. The small size of azide and alkyne tags allows the biosynthetic building blocks to which they are attached to be processed by enzymes, such as nucleotide polymerases and aminoacyl tRNA synthetases, that have poor tolerance for substrates with larger modifications such as fluorescent organic dyes.ref

The 1,2,3-triazole linkage between the azide and alkyne reaction partners (Figure 3.1.1) is extremely stable. It is not susceptible to hydrolysis, oxidation or reduction, and it survives ionization in mass spectrometry (MS) analysis. The reaction is also regiospecific, yielding exclusively 1,4-disubstituted-1,2,3-triazole linkages (Figure 3.1.1). The copper (I) catalyst is both an essential feature of the reaction and its most problematic aspect in terms of applications.ref Without the copper (I) catalyst, which accelerates the rate of the reaction by a factor of >106, the reaction is impractically slow. For convenience, copper (I) is usually prepared in situ by reduction of extraneously added copper (II) using ascorbate or TCEP (T2556, Introduction to Thiol Modification and Detection—Section 2.1). Insufficient reductive capacity can result in attenuation of in situ reactions in highly oxidizing environments.ref Copper is cytotoxic, due at least in part to its capacity for sensitizing oxidative damage to proteins and nucleic acids, and limiting applications of the azide–alkyne cyloaddition reaction in live cells. Copper/ascorbate treatment also causes extinction of R-phycoerythrin (R-PE) and GFP fluorescence.ref An excellent analysis of these considerations, together with a list of practical recommendations for their management with respect to bioconjugation applications of copper-catalyzed azide–alkyne cyloaddition chemistry, has been published by Finn and co-workers.ref Our Click-iT product portfolio, consisting of individual azide and alkyne labeling reagents and application-specific kits, is described in detail below.

Click-iT reactions
Figure 3.1.1 Click-iT copper-catalyzed azide–alkyne cycloaddition chemistry applied to detection of A) nucleic acids, B) proteins, C) carbohydrates and D) lipids. The reaction partners are A) 5-ethynyl-2'-deoxyuridine (EdU) and Alexa Fluor 488 azide, B) L-homopropargylglycine (HPG) and Alexa Fluor 488 azide, C) N-azidoacetylgalactosamine and Alexa Fluor 488 alkyne and D) 15-azidopentadecanoic acid and Alexa Fluor 488 alkyne (D). In each case, the left-hand partner is a metabolic precursor that can be incorporated into proteins and nucleic acids via de novo synthesis or post-translational modification pathways.

Figure 3.1.2 Relative size of detection molecules commonly used in cellular analysis. Because the azide and alkyne moieties can be used interchangeably to optimize labeling configurations, R1 can be either the biomolecule of interest or the detection reagent. For biotin and Alexa Fluor 488, R2 represents the biomolecule of interest.

Copper-Free Click Chemistry

Classic click reactions comprise a copper-catalyzed azide–alkyne cycloaddition to label and detect molecules of interest in cells or tissues. As described above, one drawback of this approach is that copper ions—both Cu(II) as well as Cu(I), which is produced in the presence of ascorbate or TCEP—can harm cells, reduce the fluorescence of fluorophores ref and impair protein function. The Click-iT DIBO alkyne reagents (Molecular Probes azide and alkyne derivatives—Table 3.1) are compatible with copper-free click chemistry reactions that capture or detect azide-substituted targets, including metabolic analogs of proteins and many of their posttranslationally modified forms (Figure 3.1.3).

Figure 3.1.3 Copper-free coupling of Click-iT Alexa Fluor 488 DIBO alkyne to N-azidoacetylgalactosamine.

The key to copper-free click chemistry is the DIBO (dibenzocyclooctyne) core of the Click-iT DIBO alkyne reagent. The strain in this eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts or extreme temperatures or solvents, enabling the study of the surface of live cells, and preventing copper-induced damage of fluorescent proteins such as GFP in fixed and permeabilized cells. Nine Click-iT DIBO alkyne reagents are available, including DIBO derivatives of Alexa Fluor, tetramethylrhodamine and biotin labels, as well as of reactive probes capable of modifying amine, thiol and carboxylic acid groups.  These reagents are biologically inert, and their reaction products with azides are extremely stable, containing an irreversible, covalent bond. The stability of the covalent 1,2,3-triazole linkage between the azide and alkyne reaction partners allows extensive washing to produce high signal-to-noise ratios, an important consideration when detecting intracellular targets in fixed and permeabilized cells.

Click-iT Azide and Alkyne Labeling Reagents

Although in situ labeling of biomolecules for cytochemical and proteomic analysis is perhaps the most notable application of Click-iT technology, it is by no means the only one. This specific and direct labeling methodology can also be applied to bioconjugate preparation,ref surface and particle functionalization and molecular ligations. Our Click-iT azide and alkyne labeling reagents support these applications and also provide foundational tools for developing new in situ labeling applications (Molecular Probes azide and alkyne derivatives—Table 3.1).

Azide- and Alkyne-Derivatized Dyes and Biotinylation Reagents

We offer a rich selection of azide- and alkyne-derivatized fluorescent dyes for coupling to complementary azide- and alkyne-functionalized biomolecules:

We also offer a set of Alexa Fluor, tetramethylrhodamine and biotin DIBO alkyne derivatives for copper-free click reactions with azide-modified targets:

  • Click-iT Alexa Fluor 488 DIBO alkyne (C10405)
  • Click-iT Alexa Fluor 555 DIBO alkyne (C10406)
  • Click-iT Alexa Fluor 594 DIBO alkyne (C10407)
  • Click-iT Alexa Fluor 647 DIBO alkyne (C10408)
  • Click-iT TAMRA DIBO alkyne (C10410)
  • Click-iT biotin DIBO alkyne (C10412)

Antibodies to Oregon Green 488, tetramethylrhodamine and Alexa Fluor 488 dyes (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) and Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) are available to provide signal amplification if necessary.ref The biotin azide and alkyne reagents facilitate western blotting applications and streptavidin enrichment in combination with our streptavidin or CaptAvidin agarose ref (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).

Azide- and Alkyne-Modified Nucleosides and Amino Acids

We offer the following azide- and alkyne-modified nucleosides and amino acids for Click-iT labeling protocols:

  • 5-ethynyl uridine (EU, E10345)
  • 5-ethynyl-2'-deoxyuridine (EdU; A10044, E10187; Figure 3.1.1A)
  • Click-iT AHA (L-azidohomoalanine for nascent protein synthesis, C10102)
  • Click-iT HPG (L-homopropargylglycine for nascent protein synthesis, C10186; Figure 3.1.1B)

The alkyne-modified nucleosides EdU and EU form the basis of our Click-iT cell proliferation and nascent RNA assays described below. The individual packagings of these reagents provide the larger quantities required for in vivo labeling applications.ref AHA and HPG are methionine surrogates providing nonradioactive alternatives to 35S-methionine for pulse-chase detection of protein synthesis and degradation.ref

Azide- and Alkyne-Modified Monosaccharides, Fatty Acids and Isoprenoids

The Click-iT metabolic glycoprotein labeling reagents provide biosynthetic precursors for detecting and characterizing post-translational glycosylation of proteins:ref

  • Click-iT GalNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylgalactosamine for labeling O-linked glycoproteins, C33365; Figure 3.1.1C, structure)
  • Click-iT ManNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylmannosamine for labeling sialic acid–modified glycoproteins, C33366; Figure 3.1.4)
  • Click-iT GlcNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylglucosamine for labeling O-linked N-acetylglucosamine (O-GlcNAc)–modified glycoproteins, C33367; structure)
  • Click-iT fucose alkyne (tetraacetylfucose alkyne, C10264; structure

Cultured cells are simply incubated with the modified sugars for 2–3 days or until cells reach the appropriate density. The acetyl groups improve cell permeability of the modified sugars and are removed by nonspecific intracellular esterases (Figure 3.1.4). The resulting azide- or alkyne-modified sugar is then metabolically incorporated through the permissive nature of the oligosaccharide biosynthesis pathway, yielding functionalized glycoproteins that can be chemoselectively coupled to complementary alkyne- or azide-functionalized fluorophores and biotinylation reagents for detection or affinity capture. We also offer the the Click-iT O-GlcNAc Enzymatic Labeling System for in vitro enzyme-mediated N-azidoacetylgalactosamine labeling of O-GlcNAc–modified glycoproteins ref (C33368, Detecting Protein Modifications—Section 9.4) and Click-iT Protein Analysis Detection Kits (C33370, C33371, C33372; Detecting Protein Modifications—Section 9.4) for detection of azide-functionalized glycoproteins in 1D or 2D electrophoresis gels or western blots.

Similarly, our azide-functionalized isoprenoids and fatty acids enable detection of post-translational lipidation of proteins by in-gel fluorescence scanning, fluorescence microscopy and flow cytometry.ref We offer the following azide-modified fatty acids and isoprenoids:

  • Click-iT farnesyl alcohol, azide (C10248, structure)
  • Click-iT geranylgeranyl alcohol, azide (C10249, structure)
  • Click-iT palmitic acid, azide (15-azidopentadecanoic acid, C10265; Figure 3.1.1D)
  • Click-iT myristic acid, azide (12-azidododecanoic acid, C10268)

tetraacetylated azido sugarsFigure 3.1.4 Metabolic incorporation of tetraacetylated azido sugars.

Heterobifunctional Reagents

Our collection of heterobifunctional reagents provide a means for adapting the amine- and thiol-reactive labeling chemistries described in Chapters 1 and 2 with the azide–alkyne Click-iT labeling protocols:

  • Azide succinimidyl ester (A10280, structure)
  • Alkyne succinimidyl ester (A10279, structure)
  • Azide iodoacetamide (I10188, structure)
  • Alkyne iodoacetamide (I10189, structure)

The heterobifunctional DIBO alkyne reagents provide a means of modifying amines, thiols and carboxylic acids such that they can be used for copper-free click chemistry reactions with modified azides:

  • Click-iT succinimidyl ester DIBO alkyne (C10414)
  • Click-iT maleimide DIBO alkyne (C10413)
  • Click-iT amine DIBO alkyne (C10411)

The succinimidyl ester reagents can be used for azide or alkyne functionalization of amine-containing molecules and molecular assemblies including terminally or internally modified oligonucleotides and nanoparticles.ref

Click-iT Reaction Buffers

For added convenience, we offer Click-iT Reaction Buffer Kits for protein or cell samples labeled with an azide- or alkyne-tagged biomolecule. The Click-iT Cell Reaction Buffer Kit (C10269) includes sufficient reagents to perform 50 reactions based on a 0.5 mL reaction volume for subsequent analyses by flow cytometry, fluorescence microscopy or high-content screening (HCS). The Click-iT Protein Reaction Buffer Kit (C10276) includes everything required for click coupling to functionalized proteins for subsequent standard protein biochemical analyses (e.g., western blots or mass spectrometry).

Click-iT Tools for Cell Proliferation Analysis

The Click-iT EdU cell proliferation assay provides a superior alternative to bromodeoxyuridine (BrdU) or 3H-thymidine incorporation methods for measuring new DNA synthesis.ref The alkynyl nucleoside analog EdU (5-ethynyl-2'-deoxyuridine; A10044, E10187, E10415) is incorporated into DNA during the synthesis phase (S phase) of the cell cycle and is subsequently detected by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore ref (Figure 3.1.1A). The small size of the click-coupled fluorophore compared to that of antibodies required for immunodetection of BrdU (Figure 3.1.2) enables efficient penetration of complex samples without the need for harsh cell treatment, simplifying the assay considerably. The Click-iT EdU assay protocol is compatible with both adherent cells and cell suspensions. From start to finish, the EdU detection assay is complete in as little as 90 minutes, as compared with the antibody-based BrdU method, which takes 6–24 hours to complete. In addition, the Click-iT EdU cell proliferation assay can be multiplexed with surface and intracellular marker detection using Alexa Fluor dye–labeled secondary antibodies ref (Secondary Immunoreagents—Section 7.2) (Figure 3.1.5). Although the majority of applications are in cultured mammalian cells, Click-iT EdU reagents and methods have also been successfully applied to a wide range of model organisms including:

  • Escherichia coli ref
  • Caenorhabditis elegans ref
  • Drosophila ref
  • Zebrafish ref
  • Mouse ref
  • Plants ref (alfalfa, Arabidopsis, grape, maize, rice and tobacco)
  Figure 3.1.5 Multicolor imaging with the Click-iT EdU Imaging Kits. Muntjac cells were treated with 10 µM EdU for 45 minutes. Cells were then fixed and permeabilized, and EdU that had been incorporated into newly synthesized DNA was detected by the far-red–fluorescent Click-iT EdU Alexa Fluor 647 HCS Assay Kit (C10356, C10357). Tubulin was labeled with an anti-tubulin antibody and visualized with an Alexa Fluor 350 goat anti–mouse IgG antibody (A21049). The Golgi complex was stained with the green-fluorescent Alexa Fluor 488 conjugate of lectin HPA from Helix pomatia (edible snail) (L11271), and peroxisomes were labeled with an anti-peroxisome antibody and visualized with an orange-fluorescent Alexa Fluor 555 donkey anti–rabbit IgG antibody (A31572).

Click-iT EdU Flow Cytometry Assay Kits

The Click-iT EdU Flow Cytometry Assay Kits provide all the reagents needed to perform 50 assays using 0.5 mL reaction buffer per assay, including the nucleoside analog EdU and all components for fixation, permeabilization and labeling whole blood samples, adherent cells or suspension cells:ref

  • Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (50-assay size, C10425; 100-assay size, C10420)
  • Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (50-assay size, C10424; 100-assay size, C10419)
  • Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (50-assay size, C10418)

Click-iT EdU Imaging Kits

The Click-iT EdU Imaging Kits contain all of the components needed to label and detect incorporated EdU on 50 coverslips using 0.5 mL reaction buffer per test, as well as the blue-fluorescent Hoechst 33342 nuclear stain for identification of cells irrespective of EdU incorporation status.

  • Click-iT EdU Alexa Fluor 488 Imaging Kit (C10337)
  • Click-iT EdU Alexa Fluor 555 Imaging Kit (C10338)
  • Click-iT EdU Alexa Fluor 594 Imaging Kit (C10339)
  • Click-iT EdU Alexa Fluor 647 Imaging Kit (C10340)

Click-iT EdU HCS Assay Kits

The Click-iT EdU HCS Assay Kits contain all of the materials needed to label and detect incorporated EdU in adherent cells in 96-well microplates and 100 µL reaction buffer per assay:

  • Click-iT EdU Alexa Fluor 488 HCS Assay Kit (2-plate size, C10350; 10-plate size, C10351)
  • Click-iT EdU Alexa Fluor 555 HCS Assay Kit (2-plate size, C10352; 10-plate size, C10353)
  • Click-iT EdU Alexa Fluor 594 HCS Assay Kit (2-plate size, C10354; 10-plate size, C10355)
  • Click-iT EdU Alexa Fluor 647 HCS Assay Kit (2-plate size, C10356; 10-plate size, C10357; Figure 3.1.5)

For cell registration or DNA profiling, these kits also include the blue-fluorescent HCS NuclearMask Blue stain.

Click-iT EdU Microplate Assay Kit

The Click-iT EdU Microplate Assay Kit provides a simple and rapid workflow with fewer wash steps resulting in a substantial time-savings advantage over traditional BrdU colorimetric or fluorescent cell proliferation assays. This assay uses Oregon Green 488 azide for click coupling to synthetically incorporated EdU. The signal is amplified using immunodetection of the Oregon Green 488 fluorophore by a horseradish peroxidase (HRP) conjugate of rabbit anti–fluorescein/Oregon Green dye antibody, followed by fluorogenic or chromogenic detection with our Amplex UltraRed HRP substrate. The Click-iT EdU microplate assay has been successfully tested in HeLa, A549, U2OS and A541 cells with a variety of reagents that modulate DNA synthesis, including the DNA synthesis inhibitor aphidicolin and the mitotic inhibitor paclitaxel. The Click-iT EdU Microplate Assay Kit (C10214) contains sufficient reagents for performing 400 individual assays in a 96-well plate format.

Click-iT TUNEL Assay

The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay—based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3'-OH ends of fragmented DNA—is probably the most widely used in situ test for studying apoptotic DNA fragmentation. For a sensitive and reliable TUNEL imaging assay, it is vital that the modified nucleotide is an efficient substrate for TdT. The minimally modified EdUTP nucleotide (Figure 3.1.6) used in the Click-iT TUNEL imaging assay is rapidly incorporated by TdT, allowing samples to be rapidly fixed in order to preserve late-stage apoptotic cells, thereby lessening the possibility of false-negative results due to cell detachment and subsequent loss. The enzymatically incorporated nucleotide is detected by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore. Compared with assays that use one-step incorporation of dye-modified nucleotides, the fast and sensitive Click-iT TUNEL imaging assay can detect a higher percentage of apoptotic cells under identical conditions in 2 hours or less.

The Click-iT TUNEL Imaging Assay Kits are available with a choice of azide-derivatized Alexa Fluor dyes, providing flexibility for combination with other apoptosis detection reagents (Assays for Apoptosis—Section 15.5):

  • Click-iT TUNEL Alexa Fluor 488 Imaging Assay (C10245)
  • Click-iT TUNEL Alexa Fluor 594 Imaging Assay (C10246)
  • Click-iT TUNEL Alexa Fluor 647 Imaging Assay (C10247)

The Click-iT TUNEL assay has been tested in HeLa, A549 and CHO K1 cells with a variety of reagents that induce apoptosis, including staurosporine, and multiplexed with antibody-based detection of other apoptosis biomarkers such as cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3 and phosphohistone 2B. It has also proven effective for detection of apoptosis induced by siRNA knockdown of the DEC2 transcription factor in human MCF-7 breast cancer cells.ref

EdUTP structure  
Figure 3.1.6 The EdUTP nucleotide, provided in the Click-iT TUNEL Imaging Assay Kits.

Click-iT Tools for Detecting RNA and Protein Synthesis

Click-iT RNA Kits

Click-iT RNA HCS and Click-iT RNA Imaging Assay Kits provide everything needed to detect newly synthesized RNA in adherent cells:

  • Click-iT RNA Alexa Fluor 488 HCS Assay (C10327)
  • Click-iT RNA Alexa Fluor 594 HCS Assay (C10328)
  • Click-iT RNA Alexa Fluor 488 Imaging Kit (C10329)
  • Click-iT RNA Alexa Fluor 594 Imaging Kit (C10330)

Click-iT RNA assays are ideal for imaging global RNA synthesis in multiplex analyses using traditional fluorescence microscopy or high-content screening (HCS). The Click-iT RNA assays employ the alkyne-modified nucleoside EU (5-ethynyl uridine, E10345; structure), which is supplied to cells and incorporated into nascent RNA.ref The small size of the alkyne tag enables efficient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes. Detection of incorporated EU is accomplished by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore. The multiplexing capability of the assays makes them ideal for toxicological profiling or interrogation of disease models using high-content imaging platforms.

The Click-iT RNA HCS Assay Kits (C10327, C10328) contain sufficient reagents to label and detect newly synthesized RNA in whole cells using two 96-well microplates and 50 µL reaction volumes per well. These kits also supply the blue-fluorescent HCS NuclearMask blue stain as a nuclear counterstain for cell demarcation or for DNA profiling. The Click-iT RNA Imaging Kits (C10329, C10330) contain sufficient reagents to label and detect newly synthesized RNA in whole cells using 25 coverslips and 500 µL reaction volume per well. These kits also supply the blue-fluorescent Hoechst 33342 dye as a nuclear counterstain or for DNA profiling.

The Click-iT Nascent RNA Capture Kit (C10365) enables RNA synthesized during a time window defined by administration of EU to be selectively biotinylated via click coupling of EU to biotin azide. Biotinylated RNA is then captured using streptavidin-functionalized magnetic beads for reverse transcription and subsequent analysis by DNA sequencing, PCR or microarray hybridization.

Click-iT Nascent Protein Synthesis Detection Kit

Detecting newly synthesized protein is key for researchers studying protein biosynthesis, trafficking and degradation. Click-iT AHA (L-azidohomoalanine, structure) incorporation provides a fast, sensitive and nonradioactive alternative to the traditional radioactive 35S-methionine technique for the detection of nascent protein.ref L-azidohomoalanine, an analog of L-methionine, is supplied to cultured cells and is biosynthetically incorporated into proteins. The incorporated amino acid is then detected by copper (I)–catalyzed click coupling to an alkyne-derivatized fluorophore. This two-step labeling and detection method provides detection sensitivity comparable with that obtained using the radioactive 35S-methionine method and is compatible with downstream LC-MS/MS and MALDI-MS analysis. Click-iT AHA is available as a stand-alone reagent (C10102) or in the Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay Kit (C10289), which contains Alexa Fluor 488 alkyne for detection. Click-iT AHA has proven to be a successful substitute for methionine in many cell types, including COS-7, 3T3-L1, HeLa, HEK 293 and Jurkat cells. Cellular incorporation of Click-iT AHA should be carried out in methionine-free media, as methionine is the preferred substrate for methionyl tRNA transferase, and supplemented media (i.e., methionine-free DMEM) should be used in place of HBSS to achieve greater Click-iT AHA incorporation at lower concentrations.

Click-iT Tools for Detecting Lipid Peroxidation

The Click-iT Lipid Peroxidation Imaging Kit (C10446) and stand-alone Click-iT LAA reagent (C10447) employ click chemistry and an alkyne-modified linoleic acid (linoleamide alkyne or LAA) to detect lipid peroxidation–derived protein modifications by fluorescence microscopy (Figure 3.1.7) or high-content screening. When incubated with cells, Click-iT LAA incorporates into cell membranes. Upon lipid peroxidation, LAA is oxidized and produces 9- and 13-hydroperoxy-octadecadienoic acid (HPODE). These hydroperoxides decompose to α,β-unsaturated aldehydes, which readily modify proteins at nucleophilic side chains, producing alkyne-containing proteins that can then be detected with fluorescent or biotinylated azides using click chemistry.

The Click-iT Lipid Peroxidation Imaging Kit (C10446) provides the tools needed to visualize the lipid peroxidation modifications using green-fluorescent Alexa Fluor 488 azide. To customize the click reaction, we also offer the stand-alone LAA reagent (C10447), as well as a variety of azide-modified detection reagents and the Click-iT Cell Reaction Buffer Kit (C10269, see above).

Click-iT Lipid Peroxidation Detection Kit
Figure 3.1.7
Lipid peroxidation detection in hemin-treated cells. After incubation with 50 µM LAA (provided in the Click-iT Lipid Peroxidation Kit (C10446)) in complete growth medium at 37°C for 30 min, bovine pulmonary artery endothelial (BPAE) cells were treated with 10 µM hemin to induce lipid peroxidation–derived protein modifications (right), or left untreated (left) for 2 hr at 37°C, then fixed and permeabilized. The click reaction was performed with 5 µM Alexa Fluor 488 azide for 30 min. Cells were counterstained with NucBlue Live Cell Stain (R37605) and imaged on a Zeiss Axiovert inverted microscope using a 40x objective.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A10044 icon 252.23 F,D DMSO, H2O <300   none   1
A10266 icon icon 861.04 F,D,L DMSO 493 76,000 516 pH 7  
A10267 icon icon 773.91 F,D,L DMSO 494 76,000 520 pH 7  
A10270 icon icon 948.16 F,D,L DMSO 590 105,000 617 pH 7  
A10275 icon icon 962.23 F,D,L DMSO 588 100,000 616 pH 7  
A10277 icon ~850 F,D,L DMSO 646 270,000 668 MeOH  
A10278 icon ~800 F,D,L DMSO 646 250,000 661 pH 7  
A10279 icon 225.20 F,D DMSO <300   none    
A10280 icon 388.38 F,D,L DMSO <300   none    
A20012 icon ~850 F,D,L DMSO 554 151,000 568 pH 7  
A20013 icon ~750 F,D,L DMSO 554 150,000 567 pH 7  
B10184 icon 615.79 F,D,L DMSO <300   none    
B10185 icon 528.66 F,D DMSO <300   none    
C10102 icon 258.16 F,DD DMSO <300   none    
C10186 icon 127.14 F,D DMSO <300   none    
C10248 icon 263.38 F,D,LL DMSO <300   none    
C10249 icon 331.50 F,D,LL DMSO <300   none    
C10264 icon 342.30 F,D DMSO <300   none   2
C10265 icon 283.41 F,D,L DMSO <300   none    
C10268 icon 241.33 F,D,L DMSO <300   none    
C33365 icon 430.37 F,D DMSO <300   none   3
C33366 icon 430.37 F,D DMSO <300   none   3
C33367 icon 430.37 F,D DMSO <300   none   3
E10345 icon 268.23 F,D DMSO <300   none    
I10188 icon 310.14 F,D,L DMSO <300   none   4
I10189 icon 223.01 F,D,L DMSO <300   none   4
O10180 icon icon 637.68 F,D,L DMSO 494 80,000 521 pH 9 5
O10181 icon icon 449.37 F,D,L DMSO 494 80,000 521 pH 9 5
T10182 icon icon 554.65 F,D,L DMSO 546 95,000 571 MeOH 6
T10183 icon icon 467.52 F,D,L DMSO 543 95,000 572 MeOH 6
  1. E10187 and E10415 are alternative packagings of A10044 but are otherwise identical.
  2. C10264 is deacetylated in vivo yielding the 6-ethynyl analog of fucose (MW = 174.15).
  3. C33365, C33366 and C33367 are deacetylated in vivo yielding N-azidoacetylgalactosamine, N-azidoacetylmannosamine and N-azidoacetylglucosamine (all MW = 262.22), respectively.
  4. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
  5. Absorption and fluorescence of Oregon Green 488 dyes are pH dependent only in moderately acidic solutions (pH <5).
  6. Abs and Em for tetramethylrhodamine dyes in pH 8 buffer are red-shifted approximately 8 nm compared to MeOH, with EC lower by ~10%.