Click chemistry is an uncomplicated and efficient chemical reaction set up to conjugate azide- and alkyne-modified biomolecules with or without copper as catalyst. It is sometimes called as “a chemical reaction designed to be performed by non-chemists”, which means everyone can do it easily. Since it is easy to set up, there are a lot of standard click reaction protocols available for all kind of labeling reactions depending on the targets (mRNA, Oligo, etc.) and the catalysts (CuBr, CuSO4 or heterogenous catalysts). The available wide variety of click chemistry protocols are mostly used for labeling and detection purposes. It is the best choice for these purposes because of its reproducibility, efficiency and for being a bioorthogonal reaction. Biorthogonal describes the fact that the functional groups, alkynes and azides, involved in click chemistry do not interfere or react with any other group in biomolecules. Therefore, it leads to a broad range of potential usage for click reaction protocols, including for the development of kit systems. baseclick is producing a whole series of different cell proliferation detection kits based on click chemistry with an efficient and easy click reaction protocol. The kits are specialized on the detection method of cell proliferation with its own protocol for detection by Imaging, Flow cytometry or by High-throughput screening. There is also a cell proliferation detection kit developed specificially for the detection of T-Cell proliferation with its own click reaction protocol. Another kit, the Oligo Link kit, provides only the supporting reagents and a protocol for performing click chemistry with your own alkyne and azido modified molecules.
What Is a Click Reaction Protocol?
A click reaction protocol provides you with a guideline to perform a click reaction. There are click reaction protocols optimized for their purpose of application. Exemplary applications fields can be labeling reactions, detection of (bio)molecules, crosslinking, conjugation or the preparation of molecular frameworks. To use the full potential of click chemistry, as high quantitative yields, short reaction time, tolerance of functional groups and many more, for all these applications, it is necessary to have a solid topical related click reaction protocol available. This protocol ensures the reaction works best for the molecules you plan to react to each other and provides the consistent results required for your research. These protocols usually give you step-to-step work instructions including additional steps as purification possibilities. These easy-to-follow instructions for click chemistry make it the perfect reaction for non-chemists to perform, thanks to the combination of detailed protocols and fast and simple reaction setup. Intensive training in chemistry is therefore unnecessary. These protocols have many applications, such as in kits for imaging, flow cytometry and high-throughput screening (HTS).
Applications of Kit Click Reaction Protocols
Click Reaction Protocol for Imaging Kit
Imaging applications of click reaction protocols are usually based on the conjugation of a fluorescent dye to a targeting biomolecule by click chemistry. Afterwards the samples are analyzed by imaging with a fitting filter set for the used dye. The fluorescent labeled target is now possible to localize by the signals of the conjugated fluorophore. The highly efficient protocols for click chemistry labeling enable high sensitivity, reproducibility, and a high signal to noise ratio for imaging reaction. To support these advantages, dyes with high quantum yields should be chosen which are well suited for click chemistry. To simplify the experiment, ready-to-use kits have been developed, which combine the necessary ingredients as the dye, chemicals for click reactions and sometimes even the label, which is incorporated in the biomolecule to be examined. An example for such a kit is the ClickTech EdU Cell Proliferation Kit for Imaging which combines a dye of a certain wavelength (488, 555, 594 or 647 nm), a label to be incorporated into the de novo synthesized DNA which should be targeted and all ingredients necessary for the click conjugation of the dye to the de novo synthesized DNA.
Click Reaction Protocol for Flow Cytometry
Detailed protocols play a crucial role in supporting flow cytometry-based applications. They are necessary for ensuring consistency, reproducibility, and accuracy for measuring cell parameters. These protocols are necessary to ensure standardization across experiments, to enable quantitative comparisons between fluorescence signals and to support the biological interpretation of the results. Potential applications for flow cytometry are measuring labeling intensity, incorporation (5-ethynyluridine (EdU)) or cell cycle dynamics. For the detection of incorporation of molecules as EdU, a thymidine analog which is implemented in de novo synthesized DNA, there are whole kits available including a protocol for the click reaction necessary to detect EdU. These kits include EdU, the fluorescent label, and all necessary reagents to perform the click reaction protocol for conjugation of EdU and the fluorescent label as well as reagents for cell permeabilization and fixation. Such kits are available here.
Click Reaction Protocol for High throughput Screening (HTS)
Kits designed for high throughput screening are usually designed to allow streamlining and automatization for most to all of the steps. One kit designed for high throughput screening is baseclick´s EdU Cell Proliferation Assay for High-throughput Screening. This kit utilizes EdU incorporation into de novo synthesized DNA and the detection via dye conjugation by click chemistry to the newly synthesized DNA. For this high throughput purpose, the click reaction protocol is optimized for automatization set up by reducing the laborious steps, such as custom reagent preparation and providing all ingredients ready to use for automatized pipettes or multi-channel pipettes for 96 well plates. Using click chemistry for conjugation guarantees high reaction yield under mild condition in combination with high reproducibility. Therefore, using these kits researchers can focus on data generation and analyzation instead of troubleshooting or reaction optimization.
Choosing the Right Click Reaction Protocol for Your Application
If you are still uncertain in which protocol to choose for a specific experiment, here are a few guidelines:
Before choosing a usable reaction protocol for your experiment, first check which equipment for performing and analyzing the experiments is available for kit solutions. Do you have a microscope suitable for using the imaging application or a flow cytometry for analyzing cells. If the machine is available, you also need to check if you have the correct filter set for the wavelength of the dyes available. If multiplex experiments should be performed, it also needs to be controlled that different reporters do not use similar or the same reporter wavelength to avoid any overlapping signal detections. For a single conjugation reaction, it is always usable to check our protocols for click chemistry. Here it is described for which application each variant of click chemistry is best suited. When you decide to use a certain reaction, it can be always helpful to check the literature if somebody has performed similar experiments as you plan to do. If yes, then maybe there is an optimized protocol available.
Why Partner with baseclick?
- Next-generation ligand strategies for modification of nucleic acids for e.g. specific cell targeting studies.
- Professional support and custom solution for nucleic acid bioconjugation via click chemistry.
- Expert support starting from therapeutic design until your clinical tryouts.
As a trusted partner to biopharma innovators, we help accelerate design , research and development of precision therapeutics.