Click-IT™ Geranylgeranyl Alcohol, Azide, mixed isomers - FAQs

•当铜离子在合适的化合价时，点击反应才有效。除了DIBO炔烃-叠氮化物反应外，在缺乏铜离子的条件下，叠氮化物和炔烃不会相互反应。确保在制备之后、二价铜浓度最高时，立即使用点击反应混合物。
•不要使用已经变黄的添加剂缓冲液；其必须无色状态下才有活性。
•细胞需要充分固定和通透，确保click试剂能充分接触到细胞内已掺入click底物的成分。
•部分试剂能结合到铜离子上，并减少其足以催化点击反应的有效浓度。click反应前，不要在任何缓冲液或试剂中引入任何金属螯合剂（例如EDTA、EGTA、柠檬酸盐等）。避免缓冲液和试剂中引入其他可能被氧化或还原的金属离子。进行click反应前，可能需要向细胞或组织样品加入额外的洗涤步骤。
•你可以用新鲜的实验试剂重复click反应尝试提高信号。增加click反应的时间长于30分钟不会提高低的信号。用新鲜的click反应试剂进行第二次30分钟培养在提高标记上更加有效。
•低信号同时也可能是因为EdU、EU和其他click底物的掺入水平较低。如果无法进行充分交联或使用的固定剂有误，其他掺入到细胞组件的click底物（如AHA、HPG、棕榈酸、叠氮化物等）可能会丢失。对于掺入到细胞膜或脂质的click底物，应避免使用乙醇或丙酮固定剂和透化试剂。
•在click反应时，掺入的click底物必须可被接触；相比于变性蛋白，天然蛋白中掺入的氨基酸模拟物的标记水平可能更低。
•你可能需要优化代谢标记条件，包括模拟物孵育时间和浓度。健康的、传代数不高、不太密集的细胞可能更容易掺入模拟物。如果孵育时间达到关注的细胞数目翻倍的时间，则应在多个整倍时间点设置包含额外剂量的click底物的阳性对照。

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.