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 provides 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.

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.

graph showing Click-iT® HPG assay results

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.


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, 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.
Find Click-iT tools for protein and glycan labeling

A choice of detection strategies is offered, because some biomolecules are sensitive to the amount of copper used to catalyze standard click reactions. In particular, proteins such as GFP and R-PE exhibit reduced fluorescence in a high-copper environment, and phalloidin binding is compromised. With Click-iT Plus detection reagents, the copper concentration can be optimized to preserve the signal in your fluorescent proteins and also drive the EdU click reaction.
Find Click-iT detection tools

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.

The mild click chemistry protocols do not destroy cellular epitopes and are compatible with antibody labeling and common cell or tissue staining methods.

Kits for protein analysis

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
Nascent protein synthesis
Readout
Proteins are tagged during synthesis for fluorescent labeling using "click" chemistry
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) 495/519 555/565 590/617 650/668 495/519
Signal-to-noise ratio five 

stars five stars five stars five stars five stars
Photostability four stars five stars four stars five stars five stars
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
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 5mg C10459
Farnesylated proteins Click-iT farnesyl alcohol, azide Azide 5 mg C10248
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
Product Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS 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 four stars
Multiplexing Compatible with antibody labeling and common cell or tissue staining methods
Sample type Optimized for HCS assays
Format 2 plates
Cat. No. C10428
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