Click-iT® Labeling Kits Detect Biomolecules Across Multiple Applications

Biological Discovery Is a Click Away

Accurate Detection of Cell Proliferation in Single-Pulse or Dual-Pulse Experiments

Click-iT® EdU assays are used to accurately measure proliferation through the use of a modified nucleoside, EdU (5-ethynyl-2´-deoxyuridine), which is incorporated during DNA synthesis. Although Click-iT® EdU technology has only been available since 2007, its use has already been demonstrated in a wide variety of species, including plants, bacteria, yeasts, and a broad spectrum of animals (Figure 1) [2–9].

In applications where only a single pulse is required, Click-iT® EdU assays provide not only a greatly truncated protocol compared to BrdU-based proliferation assays by eliminating the DNA denaturation step, but also an extremely bright signal without the incubations required by secondary detection methods for signal amplification. For dual-pulse applications, the use of EdU as one of the nucleoside analogs greatly simplifies the procedure, as there is no reactivity between the Click-iT® azide detection reagent and the incorporated BrdU. Moreover, several anti-BrdU antibodies, including the MoBU-1 clone, do not cross-react with EdU (Figure 2). The use of BrdU and EdU together have allowed temporal characterization of S-phase cells to investigate the relationship between cell-cycle length and neurogenesis [10].

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Figure 2. The anti-BrdU antibody clone MoBU-1 does not react with EdU. Jurkat T cells were treated with 10 μM EdU for 1 hr, the cells were fixed in ethanol, and an acid denaturation method was used before labeling with FITC conjugates of the most commonly used anti-BrdU clones: 3D4, PRB-1, and MoBU-1. Cells were analyzed with a BD™ LSR II flow cytometer using a 488 nm excitation laser, and fluorescence was collected with a 530/30 bandpass filter. Of the clones tested, only clone MoBU-1 did not react with EdU.

Mitochondrial DNA Synthesis Exposed

Changes in mitochondrial DNA (mtDNA) copy number are not only important for normal biological processes but also implicated in drug toxicity, aging, and a wide range of diseases, including cancer, diabetes, and neurodegeneration. Methods used to study the regulation of nuclear DNA replication are, in theory, applicable to mtDNA applications but have faced challenges in practice. Visualizing nascent mtDNA using the Click-iT® EdU assay requires signal amplification. Tyramide Signal Amplification (TSA®) technology (PerkinElmer) is compatible with the Click-iT® EdU assay and has been reported to increase detection sensitivity up to 100-fold compared to conventional avidin–biotinylated enzyme complex (ABC) procedures [11]. By combining the streamlined Click-iT® EdU assay with ultrasensitive TSA® detection, a superior method is established to explore the biology of mtDNA replication and turnover [12] (Figure 3), and for toxicological profiling of drugs that inhibit mtDNA synthesis.

Imaging Global RNA Synthesis

Click-iT® RNA assays are ideal for imaging global RNA synthesis in multiplex analyses using traditional fluorescence microscopy or high-content screening (HCS). The small size of the alkyne moiety (MW ~25) of EU (5-ethynyl uridine) enables efficient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes [5]. The multiplexing capability of the assays makes them ideal for toxicological profiling or interrogation of disease models using high-content imaging platforms (Figure 4). The Click-iT® RNA Imaging and Click-iT® RNA HCS kits also provide exciting new alternatives to antibody-based BrU or BrUTP assays for studying viral RNA synthesis in infected cells (Figure 5).

Study the Kinetics of Transcription and RNA Turnover

Until now, the only assay that has been widely used to generate nascent RNA for transcriptional profiling is the cumbersome nuclear run-on assay. Because transcription is measured in isolated nuclei in vitro, a major limitation of this method is that the procedure is cell-invasive and subject to experimental bias. Moreover, nuclear run-on does not allow for subsequent capture of nascent RNA. The Click-iT® Nascent RNA Capture Kit offers a simple, efficient method of capturing newly synthesized RNA for higher-resolution studies of the transcriptome that is not cell-invasive, nor does it require radioactive nucleoside analogs. The Click-iT® Nascent RNA Capture Kit enables simultaneous analysis of short-term changes in RNA synthesis and decay, and their impact on overall cellular transcript levels. To investigate the kinetics of RNA synthesis, the duration of EU treatment can be varied, followed by nascent RNA capture. mRNA turnover can then be estimated by dividing the C_t value for the nascent RNA by the C_t value for total RNA. For analysis of RNA decay, a traditional pulse-chase experiment can be performed to determine the loss of labeled RNA (Figure 6).

The Next Generation in Protein Profiling

Several recent publications [13–15] reveal that click chemistry–based tools are able to reliably and sensitively detect the presence of post-translational modifications (PTMs), delivering detection data in hours or days as opposed to the weeks or months required with radiolabeling techniques. We offer several click-modified reagents for in-depth protein analysis (Table 1). The azide- or alkyne-containing biomolecule is fed to cells or animals and becomes actively incorporated into proteins, allowing radioisotope-free detection of key PTMs and nascent protein synthesis (Figure 7).

In addition to our growing selection of labeled biomolecules, the Click-iT® O-GlcNAc Enzymatic Labeling System can be used for in vitro labeling of O-GlcNAc–modified glycoproteins. Once labeled, the modified protein is detected with the corresponding alkyne-containing dye or hapten using either the Click-iT® Cell Reaction Buffer Kit or the Click-iT® Protein Reaction Buffer Kit. With the Click-iT® Cell Reaction Buffer Kit, cells can be analyzed by fluorescence microscopy, flow cytometry, or high-content imaging and analysis. With the Click-iT® Protein Reaction Buffer Kit, proteins are compatible with common analyses including downstream LC-MS/MS and MALDI MS analysis, and detection sensitivity in 1D gels and western blots is in the low femtomole range.

Table 1. Azide- and alkyne-modified biomolecules for analysis of protein synthesis and posttranslational modifications.

Figure 7. Comparison of ³⁵S-methionine and Click-iT® AHA assay sensitivity. Cortical neurons were metabolically labeled with either Click-iT® AHA (L-azidohomoalanine) or ³⁵S-methionine (³⁵S-met) in a time series for 15–90 min. (Left) Newly synthesized proteins labeled with Click-iT® AHA were detected with the Click-iT® TAMRA Protein Analysis Kit and visualized using 532 nm excitation. (Right) Proteins labeled with ³⁵S-met were detected by storage phosphorimaging. The time course for incorporation of Click-iT® AHA vs. ³⁵S-met shows similar increases over a time frame of 15–90 min. The sensitivity of Click-iT® detection is comparable to that of ³⁵S-met; the fluorescence of the TAMRA image was quantified in the two most prominent bands, and the incorporation was linear across the entire time series (data not shown). Data contributed by Pate Skene, Duke University.

Detect DNA Fragmentation With Click-iT® TUNEL Assays

At the final stage of apoptosis, adherent cells often detach from the slide or well before they can be detected. Because cells are lost in this way, you may not be able to detect the DNA fragmentation—a hallmark and the ultimate determinant of apoptosis. Since its introduction in 1992 [16], the most widely used in situ test for studying DNA fragmentation or strand breaks is the terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay, which is based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3´-OH ends of fragmented DNA. For a reliable and reproducible TUNEL imaging assay, it is not only vital that the modified nucleotide be an acceptable substrate for TdT, but also that the detection method be sensitive and avoid any additional loss of cells from the sample. The Click-iT® TUNEL Imaging Assays meet both of these criteria. The alkyne-modified dUTP used in the Click-iT® TUNEL assay is rapidly incorporated by TdT, providing sensitive detection that allows you to fix your samples earlier and preserve late-stage apoptotic cells for imaging before they detach.

Making Biodiscovery a Snap

Life Technologies has been the leader in providing click chemistry reagents and assays for use in biological systems. This article highlights some of the applications and a few of the more than 1,400 publications to date that report the utility of click chemistry for biomolecule labeling and detection.

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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.