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Protein Synthesis and Degradation AssaysLabel nascent proteins, evaluate changes to protein expression, or study protein modification |
The independent mechanisms for the production and destruction of proteins, function together in the cell to maintain a balance, or homeostasis. The ability to label newly synthesized protein with both spatial and temporal resolution provide a way to study protein equilibrium and the consequences of its perturbation in disease and in experimental models. Click-iT Plus technology provides a way to label nascent proteins with a range of fluorescent labels, and the opportunity for temporal studies of synthesis and degradation using pulse-chase–type experiments. Proteins labeled with fluorescent markers can also be tracked through the autophagic pathway and multiplexed with LC3B, p62, or other autophagy markers to investigate the protein degradation process.
Click-iT technology can also be used to study protein modifications such as glycosylation and fatty acid attachment. The range of fluorescent labels and the choice of reaction conditions enable nascent protein labeling to be multiplexed with other cellular detection strategies, including fluorescent proteins.
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Selection guide for protein analysis
| Target | Name | Tag | Size | Cat. No. |
|---|---|---|---|---|
| Nascent proteins | Click-IT AHA (L-Azidohomoalanine) | Azide | 5 mg | C10102 |
| Click-iT HPG (L-homopropargylglycine) | Alkyne | 5 mg | C10186 | |
| OPP (O-propargyl-puromycin) | Alkyne | 5 mg | C10459 | |
| Geranylgeranylated proteins | Click-iT geranylgeranyl alcohol | Azide | 5 mg | C10249 |
| Fucosylated glycans | Click-iT fucose alkyne | Alkyne | 5 mg | C10264 |
| Palmitylated proteins | Click-iT palmitic acid, azide | Azide | 1 mg | C10265 |
| Myristoylated proteins | Click-iT myristic acid, azide | Azide | 1 mg | C10268 |
| O-Linked glycoproteins | Click-iT GalNAz (tetraacetylated N-azidoacetylgalactosamine) | Azide | 1 each | C33365 |
| Sialic acid-modified glycoproteins | Click-iT ManNAz (tetraacetylated N-azidoacetyl-D-mannosamine | Azide | 1 each | C33366 |
| O-GlcNAz-modified glycoproteins | Click-iT GlcNAz (tetraacetylated N-azidoacetylglucosamine) | Azide | 1 each | C33367 |
Click-iT AHA kits and tools
The ability to detect and characterize newly synthesized proteins or changes in protein expression resulting from disease, drug treatment, or environmental change is an important parameter in cytotoxicity measurements. Click-iT AHA technology provides a fast, sensitive, non-toxic, and non-radioactive method for detection of nascent protein synthesis utilizing fluorescence microscopy, flow cytometry, and high-content screening (HCS). Click-iT AHA can also be used for nascent protein capture and with protein analysis detection kits in electrophoresis gels and western blots.
- Optimized kits for HCS
- Flexible tools for multiple platforms
- Multiplex with Click-iT HPG
Figure 1. Dose-response curves performed in duplicate. U2OS cells were treated in L-methionine-free media and 50 μM Click-iT AHA for 30 minutes with anisomycin (17 pM–1 μM), cycloheximide (85 pM–5 μM), or puromycin (1.7 nM–100 μM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor® 488 alkyne. Imaging and analysis was performed using the Thermo Fisher Scientific Cellomics® ArrayScan® VTI platform.
Tracking post-translational modifications
Click-iT tools for labeling nascent protein synthesis include labels for detecting a range of post-translational modifications, including fatty acid attachment, and glycosylation. These azide-containing biomolecules are fed to cells or animals and actively incorporated into proteins.
Figure 2. Tetrahymena pyriformis staining using Click-iT GalNAz glycoprotein labeling reagent. Following fixation and permeabilization using the Image-iT Fixation/Permeabilization Kit, EdU-incorporated DNA was labeled with Alexa Fluor 488 azide and GalNAz-incorporated cellular components with Alexa Fluor 555 alkyne.
| Product | Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit | Click-iT HPG Alexa Fluor 594 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit | ||
|---|---|---|---|---|---|---|---|
| Target | |||||||
| Readout | |||||||
| Fluorescent label | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 | ||
| Standard filter set | FITC | Texas Red | FITC | Texas Red | Cy®5 | ||
| Ex/Em (nm) | 495/519 | 590/617 | 495/519 | 590/617 | 650/670 | ||
| Signal-to-noise ratio |  | |  | | | ||
| Photostability |  | | | | | ||
| Multiplexing | Compatible with antibody labeling and common cell or tissue staining methods | Retains signal from fluorescent proteins, suitable for in vivo labeling | |||||
| Sample type | Optimized for cultured cells | No-wash protocol; reagents can be added directly to culture medium | |||||
| Format | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | ||
| Cat. No. | C10428 | C10429 | C10456 | C10457 | C10458 | ||
Click-iT HPG kits for protein homeostasis
Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, non-toxic, and non-radioactive methods for the detection of nascent protein synthesis utilizing fluorescence microscopy, high-content imaging, or flow cytometry. L-homopropargylglycine (HPG), an amino acid analog of methionine that contains an alkyne moiety, is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of an Alexa Fluor azide leads to a chemoselective "click" reaction between the fluorescent azide and the alkyne, allowing the modified proteins to be detected by imaged-based analysis.
- Optimized for cultured cells
- Quantitative measurement of protein synthesis
- Multiplex with Click-iT AHA
The Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit can be used in conjunction with the Click-iT AHA with Alexa Fluor 594 Alkyne for spatial or temporal determination of differences in nascent protein synthesis.
Figure 3. Using Click-iT HPG to monitor the inhibition of protein synthesis with structurally unrelated molecules. The dose-dependent decrease was monitored using the Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit using 2 drugs and 3 different cell types.
Click-iT OPP for fast and sensitive detection
Click-iT Plus OPP Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, and non-radioactive methods for the detection of protein synthesis using fluorescence microscopy, high-content imaging, or flow cytometry. In this assay, O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in a complete methionine-containing medium, and then fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly specific, mild click reaction.
- Works in complete media
- Retains signal from fluorescent proteins
- Suitable for in vivo labeling
Figure 4. The Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit was used to label nascent protein synthesis (shown as blue punctate fluorescence within the nuclei). Talin-GFP fusion protein is shown in green, and F-actin staining is shown in red.
| Product | Click-iT AHA Alexa Fluor 488 Protein Synthesis Assay Kit |
|---|---|
| Target | Nascent protein synthesis |
| Readout | Proteins are tagged during synthesis for fluorescent labeling using "click" chemistry |
| Fluorescent label | Alexa Fluor 488 |
| Standard filter set | FITC |
| Ex/Em (nm) | 495/519 |
| Signal/Noise |  |
| Photostability |  |
| Multiplexing | Compatible with antibody labeling and common cell or tissue staining methods |
| Sample type | Optimized for HCS assays |
| Format | 2 plates |
| Cat. No. | C10289 |
Click-iT AHA kit for protein synthesis
The Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay provides a fast, sensitive, non-toxic, and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy and high-throughput imaging. Click-iT AHA is added to cultured cells and the amino acid is incorporated into proteins during active protein synthesis. Detection of the incorporated amino acid utilizes a chemoselective ligation or click reaction between an azide and alkyne, where the azido-modified protein is detected with an Alexa Fluor 488 alkyne.
Click image to enlarge
Figure 5. Dose response curves performed in duplicate. U-2 OS cells were treated in L-methionine-free media and 50 μM Click-iT AHA for 30 minutes with anisomycin (17 pM–1 μM), cycloheximide (85 pM–5 μM), or puromycin (1.7 nM–100 μM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor 488 alkyne. Imaging and analysis were performed using the Thermo Fisher Scientific Cellomics ArrayScan VTI platform.
| Product | Click-iT Protein Enrichment Kit, for click chemistry capture of azide-modified proteins |
|---|---|
| Target | Nascent protein synthesis |
| Readout | Proteins are tagged during synthesis for capture using "click" chemistry |
| Format | Spin column |
| Cat. No. | C10416 |
| Target | Name | Tag | Size | Cat. No. |
|---|---|---|---|---|
| Nascent proteins | Click-IT AHA (L-Azidohomoalanine) | Azide | 5 mg | C10102 |
| Click-iT HPG (L-homopropargylglycine) | Alkyne | 5 mg | C10186 | |
| OPP (O-propargyl-puromycin) | Alkyne | 5 mg | C10459 | |
| Geranylgeranylated proteins | Click-iT geranylgeranyl alcohol | Azide | 5 mg | C10249 |
| Fucosylated glycans | Click-iT fucose alkyne | Alkyne | 5 mg | C10264 |
| Palmitylated proteins | Click-iT palmitic acid, azide | Azide | 1 mg | C10265 |
| Myristoylated proteins | Click-iT myristic acid, azide | Azide | 1 mg | C10268 |
| O-Linked glycoproteins | Click-iT GalNAz (tetraacetylated N-azidoacetylgalactosamine) | Azide | 1 each | C33365 |
| Sialic acid-modified glycoproteins | Click-iT ManNAz (tetraacetylated N-azidoacetyl-D-mannosamine | Azide | 1 each | C33366 |
| O-GlcNAz-modified glycoproteins | Click-iT GlcNAz (tetraacetylated N-azidoacetylglucosamine) | Azide | 1 each | C33367 |
Click-iT AHA kits and tools
The ability to detect and characterize newly synthesized proteins or changes in protein expression resulting from disease, drug treatment, or environmental change is an important parameter in cytotoxicity measurements. Click-iT AHA technology provides a fast, sensitive, non-toxic, and non-radioactive method for detection of nascent protein synthesis utilizing fluorescence microscopy, flow cytometry, and high-content screening (HCS). Click-iT AHA can also be used for nascent protein capture and with protein analysis detection kits in electrophoresis gels and western blots.
- Optimized kits for HCS
- Flexible tools for multiple platforms
- Multiplex with Click-iT HPG
Figure 1. Dose-response curves performed in duplicate. U2OS cells were treated in L-methionine-free media and 50 μM Click-iT AHA for 30 minutes with anisomycin (17 pM–1 μM), cycloheximide (85 pM–5 μM), or puromycin (1.7 nM–100 μM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor® 488 alkyne. Imaging and analysis was performed using the Thermo Fisher Scientific Cellomics® ArrayScan® VTI platform.
Tracking post-translational modifications
Click-iT tools for labeling nascent protein synthesis include labels for detecting a range of post-translational modifications, including fatty acid attachment, and glycosylation. These azide-containing biomolecules are fed to cells or animals and actively incorporated into proteins.
Figure 2. Tetrahymena pyriformis staining using Click-iT GalNAz glycoprotein labeling reagent. Following fixation and permeabilization using the Image-iT Fixation/Permeabilization Kit, EdU-incorporated DNA was labeled with Alexa Fluor 488 azide and GalNAz-incorporated cellular components with Alexa Fluor 555 alkyne.
| Product | Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit | Click-iT HPG Alexa Fluor 594 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit | Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit | ||
|---|---|---|---|---|---|---|---|
| Target | |||||||
| Readout | |||||||
| Fluorescent label | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 | ||
| Standard filter set | FITC | Texas Red | FITC | Texas Red | Cy®5 | ||
| Ex/Em (nm) | 495/519 | 590/617 | 495/519 | 590/617 | 650/670 | ||
| Signal-to-noise ratio | | | | | | ||
| Photostability | | | | | | ||
| Multiplexing | Compatible with antibody labeling and common cell or tissue staining methods | Retains signal from fluorescent proteins, suitable for in vivo labeling | |||||
| Sample type | Optimized for cultured cells | No-wash protocol; reagents can be added directly to culture medium | |||||
| Format | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | 25 cover slips/ 2 plates | ||
| Cat. No. | C10428 | C10429 | C10456 | C10457 | C10458 | ||
Click-iT HPG kits for protein homeostasis
Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, non-toxic, and non-radioactive methods for the detection of nascent protein synthesis utilizing fluorescence microscopy, high-content imaging, or flow cytometry. L-homopropargylglycine (HPG), an amino acid analog of methionine that contains an alkyne moiety, is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of an Alexa Fluor azide leads to a chemoselective "click" reaction between the fluorescent azide and the alkyne, allowing the modified proteins to be detected by imaged-based analysis.
- Optimized for cultured cells
- Quantitative measurement of protein synthesis
- Multiplex with Click-iT AHA
The Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit can be used in conjunction with the Click-iT AHA with Alexa Fluor 594 Alkyne for spatial or temporal determination of differences in nascent protein synthesis.
Figure 3. Using Click-iT HPG to monitor the inhibition of protein synthesis with structurally unrelated molecules. The dose-dependent decrease was monitored using the Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit using 2 drugs and 3 different cell types.
Click-iT OPP for fast and sensitive detection
Click-iT Plus OPP Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, and non-radioactive methods for the detection of protein synthesis using fluorescence microscopy, high-content imaging, or flow cytometry. In this assay, O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in a complete methionine-containing medium, and then fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly specific, mild click reaction.
- Works in complete media
- Retains signal from fluorescent proteins
- Suitable for in vivo labeling
Figure 4. The Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit was used to label nascent protein synthesis (shown as blue punctate fluorescence within the nuclei). Talin-GFP fusion protein is shown in green, and F-actin staining is shown in red.
| Product | Click-iT AHA Alexa Fluor 488 Protein Synthesis Assay Kit |
|---|---|
| Target | Nascent protein synthesis |
| Readout | Proteins are tagged during synthesis for fluorescent labeling using "click" chemistry |
| Fluorescent label | Alexa Fluor 488 |
| Standard filter set | FITC |
| Ex/Em (nm) | 495/519 |
| Signal/Noise | |
| Photostability | |
| Multiplexing | Compatible with antibody labeling and common cell or tissue staining methods |
| Sample type | Optimized for HCS assays |
| Format | 2 plates |
| Cat. No. | C10289 |
Click-iT AHA kit for protein synthesis
The Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay provides a fast, sensitive, non-toxic, and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy and high-throughput imaging. Click-iT AHA is added to cultured cells and the amino acid is incorporated into proteins during active protein synthesis. Detection of the incorporated amino acid utilizes a chemoselective ligation or click reaction between an azide and alkyne, where the azido-modified protein is detected with an Alexa Fluor 488 alkyne.
Click image to enlarge
Figure 5. Dose response curves performed in duplicate. U-2 OS cells were treated in L-methionine-free media and 50 μM Click-iT AHA for 30 minutes with anisomycin (17 pM–1 μM), cycloheximide (85 pM–5 μM), or puromycin (1.7 nM–100 μM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor 488 alkyne. Imaging and analysis were performed using the Thermo Fisher Scientific Cellomics ArrayScan VTI platform.
| Product | Click-iT Protein Enrichment Kit, for click chemistry capture of azide-modified proteins |
|---|---|
| Target | Nascent protein synthesis |
| Readout | Proteins are tagged during synthesis for capture using "click" chemistry |
| Format | Spin column |
| Cat. No. | C10416 |
Buffers for Click-iT reactions
Click-iT Reaction Buffer Kits are optimized for proteins or cell samples that are labeled with an azide- or alkyne-tagged biomolecule.
| Product | Application |
|---|---|
| Click-iT Cell Reaction Buffer Kit | Perform 50 reactions based on a 0.5 mL reaction volume for subsequent analysis for flow cytometry, fluorescence microscopy, or high-content screening (HCS) |
| Click-iT Protein Reaction Buffer Kit | Perform the click reaction of proteins for subsequent standard protein biochemical analysis (e.g., western blots, mass spectrometry). |
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