Click-IT™ AHA (L-Azidohomoalanine) - Thermo Fisher Scientific

Click-IT™ AHA (L-Azidohomoalanine)Citations & References (30)Invitrogen™Click-IT™ AHA (L-Azidohomoalanine)Click-iT® AHA (L-azidohomoalaine) provides a fast, sensitive, non-toxic and most importantly non-radioactive alternative to the traditional radioactive technique, 35S-methionine forRead moreHave Questions?

Catalog Number	Quantity
C10102	5 mg

Catalog number C10102Price (USD) - Quantity:5 mgClick-iT® AHA (L-azidohomoalaine) provides a fast, sensitive, non-toxic and most importantly non-radioactive alternative to the traditional radioactive technique, 35S-methionine for the detection of nascent protein. AHA is an amino acid analog that contains a very small modification, specifically an azido moiety that can be fed to cultured cells and incorporated into proteins during active protein synthesis. Detection utilizes the chemoselective ligation or “click " reaction between an azide and an alkyne where the azido modified protein is detected with one of the Click-iT® Protein Analysis Detection Kits containing either TAMRA, Dapoxyl®, or biotin alkyne. Detection sensitivity with these reagents in 1-D gels and Western blots is in the low femtomole range and compatible with downstream LC-MS⁄MS and MALDI MS analysis or Multiplexed Proteomics® reagents for differential analyses of newly synthesized proteins together with total glycoproteins, total phosphoproteins or total protein.For Research Use Only. Not for use in diagnostic procedures.SpecificationsDetection MethodBiotin-based, FluorescenceGreen FeaturesLess hazardousLabeling MethodMetabolic LabelingProduct LineClick-iT, Molecular ProbesProduct TypeAHAQuantity5 mgShipping ConditionRoom TemperatureLabeling TargetProteinsLabel or DyeAlexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Biotin, Oregon Green 488, TMR (Tetramethylrhodamine)Unit SizeEachContents & StorageStore in freezer -5°C to -30°C.

Frequently asked questions (FAQs)

I am using Click-iT AHA (L-azidohomoalanine) kit to label nascent proteins in live cells, then detecting with TAMRA alkyne after fixation and permeabilization and a click reaction. But I'm seeing nucleolar labeling in the cells. It this expected?

Yes. All proteins synthesized during the time the AHA is present will be detected, and they may be all over the cell. Our imaging shows strong labeling in the nucleoli and cytoplasm, as well as nuclear labeling.Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I am observing no signal or very low signal for my click-labeled samples. What can I do to improve the signal?

The click reaction is only effective when copper is in the appropriate valency. Except for the DIBO alkyne-azide reaction, azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest. Do not use additive buffer that has turned yellow; it must be colorless to be active. Cells need to be adequately fixed and permeabilized for the click reagents to have access to intracellular components that have incorporated the click substrate(s). Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be oxidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction. You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling. Low signal can also be due to low incorporation of EdU, EU, or other click substrates. Other click substrates (e.g., AHA, HPG, palmitic acid, azide, etc.) incorporated into cellular components may have been lost if not adequately cross-linked in place or if the wrong fixative was used. For click substrates that are incorporated into the membrane or lipids, you should avoid the use of alcohol or acetone fixatives and permeabilizing agents. The incorporated click substrate must be accessible at the time of the click reaction; labeling of incorporated amino acid analogs may be lower in native proteins relative to denatured proteins. You may need to optimize the metabolic labeling conditions including analog incubation time or concentration. Cells that are healthy, not too high of a passage number and not too crowded may incorporate the analog better. You may create a positive control by including extra doses of the click substrate during multiple time points during an incubation time that spans or closely spans the doubling time of the cell type of interest. Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Citations & References (30)

Citations & ReferencesAbstractSelective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Authors:Schuman EMJournal:Proceedings of the National Academy of Sciences of the United States of AmericaPubMed ID:16769897In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We ... MoreIn situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.Authors:Schuman EMJournal:Nature neurosciencePubMed ID:20543841Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging ... MoreTwo-color labeling of temporally defined protein populations in mammalian cells.Authors:Tirrell DAJournal:Bioorganic & medicinal chemistry lettersPubMed ID:18774715The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in ... MoreFluorescence visualization of newly synthesized proteins in mammalian cells.Authors:Tirrell DAJournal:Angewandte Chemie (International ed. in English)PubMed ID:17036290Noncanonical amino acid tagging enables the selective fluorescent visualization of newly synthesized proteins in mammalian cells (see the picture). Susceptibility to tagging is determined by the spatial and temporal character of the protein synthesis, thus providing a complement to methods which identify relevant members of the proteome. ... MoreSpatial coupling of mTOR and autophagy augments secretory phenotypes.Authors:Narita M., et alJournal:Science (New York, N.Y.)PubMed ID:21512002Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the ... MoreView all Click-IT™ AHA (L-Azidohomoalanine) citations