Click-iT® Detection of Protein Synthesis & Post Translational Modification
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Click-iT® Tools for Protein Post-Translational Modification (PTM) Analysis
Radioactivity and Western Blots Tools
Traditional tools for protein analyses include radioactivity and western blots. Although very specific and sensitive, radioactive methods involve costly handling and disposal, lengthy incubations and cannot be multiplexed-single parameter results only. With western blots, suitable antibodies do not always exist and sometimes even if they do, they may not be very sensitive or reliable.
Click Chemistry Reaction between Azide-Alkyne Tag Pair
The powerful new click chemistry reaction between the azide-alkyne tag pair enables one to label molecules of interest in complex biological samples similar to a radiolabeled compound, then robustly and reliably detect those molecules with unprecedented sensitivity and extremely low background. Detection of post-translational modifications and new protein synthesis can now be performed with colorimetric, chemiluminescent or fluorescence and more importantly together with other biomarkers of interest for truly in-depth and content-rich results - all with a simple, two-step procedure.
Traditional tools for protein analyses include radioactivity and western blots. Although very specific and sensitive, radioactive methods involve costly handling and disposal, lengthy incubations and cannot be multiplexed-single parameter results only. With western blots, suitable antibodies do not always exist and sometimes even if they do, they may not be very sensitive or reliable.
Click Chemistry Reaction between Azide-Alkyne Tag Pair
The powerful new click chemistry reaction between the azide-alkyne tag pair enables one to label molecules of interest in complex biological samples similar to a radiolabeled compound, then robustly and reliably detect those molecules with unprecedented sensitivity and extremely low background. Detection of post-translational modifications and new protein synthesis can now be performed with colorimetric, chemiluminescent or fluorescence and more importantly together with other biomarkers of interest for truly in-depth and content-rich results - all with a simple, two-step procedure.
 | STEP 1. Azide- or alkyne-containing biomolecule becomes actively incorporated into proteins. This allows radioisotope-free detection of key PTMs and nascent protein synthesis (Table 1 , Figure 1). |
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Figure 1. Metabolic incorporation of tetraacetylated azido-mannosamine (Ac4ManNAz).
| | STEP 2. Modified protein is detected with the corresponding alkyne-containing dye or hapten (Table 2, Figure 2) using either the Click-iT® Cell Reaction Buffer Kit or the Click-iT® Protein Reaction Buffer Kit. |
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Figure 2. Click azide/alkyne reaction. The azide and alkyne moieties are interchangeable, whereupon the molecule can be labeled with an alkyne or azide and be detected with the corresponding a fluorophore- or hapten-azide or -alkyne.
Performing Click-iT® Reactions
- With the Click-iT® Cell Reaction Buffer Kit, analyze cells by fluorescence microscopy, flow cytometry, or high-content imaging and analysis, using detection reagents listed in (Table 2).
- With the Click-iT® Protein Reaction Buffer Kit, proteins are detected with either biotin or tetramethylrhodamine and compatible with common analyses, including downstream LC-MS/MS and MALDI MS. Detection sensitivity is in the low-femtomole range.
Table 1. Click-iT® Metabolic Labeling Reagents for Proteins
| Application | Compound | Azide or alkyne | Cat. no. |
|---|---|---|---|
| Monitor nascent protein synthesis or inhibition | Click-iT® AHA (L-azidohomoalanine) | azide | C10102 |
| Click-iT® HPG (L-homopropargylglycine) | alkyne | C10186 | |
| Identification of isoprenylated proteins | Click-iT® farnesyl alcohol, azide | azide | C10248 |
| Click-iT® geranylgeranyl alcohol, azide | azide | C10249 | |
| Identification of fucoslyated proteins | Click-iT® fucose alkyne (tetraacetylfucose alkyne) | alkyne | C10264 |
| Identification of protein fatty acylation | Click-iT® palmitic acid, azide | azide | C10265 |
| Click-iT® myristic acid, azide | azide | C10268 | |
| Identification of O-linked glycoproteins, including O-GlcNAc | Click-iT® GalNAz (tetraacetylated N-azidoacetylgalactosamine) | azide | C33365 |
| Identification of sialic acid-modified glycoproteins | Click-iT® ManNAz (tetraacetylated N-azidoacetylmannosamine) | azide | C33366 |
| Identification of O-GlcNAc-modified glycoproteins | Click-iT® ManNAz (tetraacetylated N-azidoacetylglucoosamine) | azide | C33367 |
Table 2. Azide-or Alkyne-Modified Fluorophores and Haptens
| Label | Ex/Em* | Azide or alkyne | Cat. no. | Use |
|---|---|---|---|---|
| Alexa Fluor® 488 | 495/519 | azide | A10266 | Fluorescent dye or hapten** |
| alkyne | A10267 | |||
| Alexa Fluor® 555 | 555/565 | azide | A20012 | Fluorescent dye |
| alkyne | A20013 | |||
| Alexa Fluor® 594 | 590/617 | azide | A10270 | Fluorescent dye |
| alkyne | A10275 | |||
| Alexa Fluor® 647 | 650/668 | azide | A10277 | Fluorescent dye |
| alkyne | A10278 | |||
| Biotin † | NA | azide | B10184 | Hapten |
| alkyne | B10185 | |||
| Oregon Green® 488 | 496/524 | azide | O10180 | Fluorescent dye or hapten** |
| alkyne | O10181 | |||
| Tetramethylrhodamine (TAMRA) | 555/580 | azide | T10182 | Fluorescent dye or hapten** |
| alkyne | T10183 | |||
| *Absorption and fluorescence emission maxima, in nm. † Requires streptavidin for detection. **As hapten, use with anti-dye antibody. | ||||
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Find Your Click-iT® Detection Assay
With its equally small “footprint,” the Click-iT® detection molecule can easily penetrate complex samples for in-depth analysis of key biological processes, including assays for:
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Cutting-edge Click-iT® Citations
- EdU, a new Thymidine Analogue for Labeling Proliferating Cells in the Nervous System—J. Neurosci Meth. 177, 122-130 (2009)
- Exploring RNA Transcription and turnover in-vivo by using Click Chemistry—PNAS 105, 15779-15784 (2008)
- Protein Synthesis in Distal Axons is not required for Growth Cone Responses to Guidance Cues—J. Neurosci. 29, 638-652 (2009)
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