Protein Synthesis and Degradation Assays

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 provide 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.

Selection guide for protein analysis

TargetNameTagSizeCat. No.
Nascent proteinsClick-IT AHA (L-Azidohomoalanine)Azide5 mgC10102
Click-iT HPG (L-homopropargylglycine)Alkyne5 mgC10186
OPP (O-propargyl-puromycin)Alkyne5mgC10459
Geranylgeranylated proteinsClick-iT geranylgeranyl alcoholAzide5 mgC10249
Fucosylated glycansClick-iT fucose alkyneAlkyne5 mgC10264
Palmitylated proteinsClick-iT palmitic acid, azideAzide1 mgC10265
Myristoylated proteinsClick-iT myristic acid, azideAzide1 mgC10268
O-Linked glycoproteinsClick-iT GalNAz (tetraacetylated N-azidoacetylgalactosamine)Azide1 eachC33365
Sialic acid-modified glycoproteinsClick-iT ManNAz (tetraacetylated N-azidoacetyl-D-mannosamineAzide1 eachC33366
O-GlcNAz-modified glycoproteinsClick-iT GlcNAz (tetraacetylated N-azidoacetylglucosamine)Azide1 eachC33367

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 provide a fast, sensitive, nontoxic, and nonradioactive 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
Dose response curves

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 posttranslational modifications

Click-iT tools for labeling nascent protein synthesis include labels for detecting a range of posttranslational modifications, including fatty acid attachment and glycosylation. These azide-containing biomolecules are fed to cells or animals and actively incorporated into proteins.

Tetrahymena pyriformis staining

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.

ProductClick-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
Nascent protein synthesis
Readout
Proteins are tagged during synthesis for fluorescent labeling using "click" chemistry
Fluorescent labelAlexa Fluor 488Alexa Fluor 594Alexa Fluor 488Alexa Fluor 594 Alexa Fluor 647
Standard filter setFITCTexas RedFITCTexas RedCy®5
Ex/Em (nm)495/519590/617495/519590/617650/670
Signal-to-noise ratiofive 

starsfive starsfive starsfive starsfive stars
Photostabilityfour starsfive starsfour starsfive starsfive stars
MultiplexingCompatible with antibody labeling and common cell or tissue staining methodsRetains signal from fluorescent proteins, suitable for in vivo labeling
Sample typeOptimized for cultured cellsNo-wash protocol; reagents can be added directly to culture medium
Format25 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.C10428C10429C10456C10457C10458

Click-iT HPG kits for protein homeostasis

Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, nontoxic, and nonradioactive 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.

Click-iT-HPG-AF488---Figure-1

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 two drugs and three 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 nonradioactive 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
nascent protein synthesis

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.

ProductClick-iT AHA Alexa Fluor 488 Protein Synthesis Assay Kit
TargetNascent protein synthesis
ReadoutProteins are tagged during synthesis for fluorescent labeling using "click" chemistry
Fluorescent labelAlexa Fluor 488
Standard filter setFITC
Ex/Em (nm)495/519
Signal/Noise
Photostabilityfour stars
MultiplexingCompatible with antibody labeling and common cell or tissue staining methods
Sample typeOptimized for HCS assays
Format2 plates
Cat. No.C10289

Click-iT AHA kit for protein synthesis

The Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay provide 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.

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.
ProductClick-iT Protein Enrichment Kit, for click chemistry capture of azide-modified proteins
TargetNascent protein synthesis
ReadoutProteins are tagged during synthesis for capture using "click" chemistry
FormatSpin column
Cat. No.C10416
TargetNameTagSizeCat. No.
Nascent proteinsClick-IT AHA (L-Azidohomoalanine)Azide5 mgC10102
Click-iT HPG (L-homopropargylglycine)Alkyne5 mgC10186
OPP (O-propargyl-puromycin)Alkyne5mgC10459
Geranylgeranylated proteinsClick-iT geranylgeranyl alcoholAzide5 mgC10249
Fucosylated glycansClick-iT fucose alkyneAlkyne5 mgC10264
Palmitylated proteinsClick-iT palmitic acid, azideAzide1 mgC10265
Myristoylated proteinsClick-iT myristic acid, azideAzide1 mgC10268
O-Linked glycoproteinsClick-iT GalNAz (tetraacetylated N-azidoacetylgalactosamine)Azide1 eachC33365
Sialic acid-modified glycoproteinsClick-iT ManNAz (tetraacetylated N-azidoacetyl-D-mannosamineAzide1 eachC33366
O-GlcNAz-modified glycoproteinsClick-iT GlcNAz (tetraacetylated N-azidoacetylglucosamine)Azide1 eachC33367

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 provide a fast, sensitive, nontoxic, and nonradioactive 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
Dose response curves

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 posttranslational modifications

Click-iT tools for labeling nascent protein synthesis include labels for detecting a range of posttranslational modifications, including fatty acid attachment and glycosylation. These azide-containing biomolecules are fed to cells or animals and actively incorporated into proteins.

Tetrahymena pyriformis staining

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.

ProductClick-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
Nascent protein synthesis
Readout
Proteins are tagged during synthesis for fluorescent labeling using "click" chemistry
Fluorescent labelAlexa Fluor 488Alexa Fluor 594Alexa Fluor 488Alexa Fluor 594 Alexa Fluor 647
Standard filter setFITCTexas RedFITCTexas RedCy®5
Ex/Em (nm)495/519590/617495/519590/617650/670
Signal-to-noise ratiofive 

starsfive starsfive starsfive starsfive stars
Photostabilityfour starsfive starsfour starsfive starsfive stars
MultiplexingCompatible with antibody labeling and common cell or tissue staining methodsRetains signal from fluorescent proteins, suitable for in vivo labeling
Sample typeOptimized for cultured cellsNo-wash protocol; reagents can be added directly to culture medium
Format25 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.C10428C10429C10456C10457C10458

Click-iT HPG kits for protein homeostasis

Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits provide fast, sensitive, nontoxic, and nonradioactive 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.

Click-iT-HPG-AF488---Figure-1

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 two drugs and three 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 nonradioactive 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
nascent protein synthesis

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.

ProductClick-iT AHA Alexa Fluor 488 Protein Synthesis Assay Kit
TargetNascent protein synthesis
ReadoutProteins are tagged during synthesis for fluorescent labeling using "click" chemistry
Fluorescent labelAlexa Fluor 488
Standard filter setFITC
Ex/Em (nm)495/519
Signal/Noise
Photostabilityfour stars
MultiplexingCompatible with antibody labeling and common cell or tissue staining methods
Sample typeOptimized for HCS assays
Format2 plates
Cat. No.C10289

Click-iT AHA kit for protein synthesis

The Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay provide 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.

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.
ProductClick-iT Protein Enrichment Kit, for click chemistry capture of azide-modified proteins
TargetNascent protein synthesis
ReadoutProteins are tagged during synthesis for capture using "click" chemistry
FormatSpin 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.

ProductApplication
Click-iT Cell Reaction Buffer KitPerform 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 KitPerform the click reaction of proteins for subsequent standard protein biochemical analysis (e.g., western blots, mass spectrometry).

For Research Use Only. Not for use in diagnostic procedures.