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.
