Click-iT® Detection of Protein Synthesis and Protein Post-Translational Modification

Click-iT® Detection of Protein Synthesis & Post Translational Modification

Open the door to a radioactivity-free lab
Click chemistry detects key post-translational modifications (PTM)
Data delivery in hours to days
Experience the ultimate in flexible detection-with cells, gels, blots or even mass spectrometry analyses

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.

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).

(click to enlarge)

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
Monitor nascent protein synthesis or inhibition	Click-iT® HPG (L-homopropargylglycine)	alkyne	C10186
Identification of isoprenylated proteins	Click-iT® farnesyl alcohol, azide	azide	C10248
Identification of isoprenylated proteins	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
Identification of protein fatty acylation	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**
Alexa Fluor® 488	495/519	alkyne	A10267	Fluorescent dye or hapten**
Alexa Fluor® 555	555/565	azide	A20012	Fluorescent dye
Alexa Fluor® 555	555/565	alkyne	A20013	Fluorescent dye
Alexa Fluor® 594	590/617	azide	A10270	Fluorescent dye
Alexa Fluor® 594	590/617	alkyne	A10275	Fluorescent dye
Alexa Fluor® 647	650/668	azide	A10277	Fluorescent dye
Alexa Fluor® 647	650/668	alkyne	A10278	Fluorescent dye
Biotin †	NA	azide	B10184	Hapten
Biotin †	NA	alkyne	B10185	Hapten
Oregon Green® 488	496/524	azide	O10180	Fluorescent dye or hapten**
Oregon Green® 488	496/524	alkyne	O10181	Fluorescent dye or hapten**
Tetramethylrhodamine (TAMRA)	555/580	azide	T10182	Fluorescent dye or hapten**
Tetramethylrhodamine (TAMRA)	555/580	alkyne	T10183	Fluorescent dye or hapten**
Absorption and fluorescence emission maxima, in nm. † Requires streptavidin for detection. *As hapten, use with anti-dye antibody.

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: