PA Janelia Fluor® 646, SE

Janelia Fluor fluorescent dyes do not inherently possess HaloTag-binding activity; however, reactive groups such as NHS esters serve as reaction sites for covalent coupling-via chemical synthesis-to an amino-functionalized HaloTag chloroalkane ligand scaffold, thereby yielding a fluorescent HaloTag ligand.
Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
Preparation of stock solution
Solvent: Dissolved in high-quality anhydrous DMSO, with a concentration of 1-10 mM.
Dissolution aid: To ensure complete dissolution, if necessary, the test tube can be heated to 37°C and then agitated in an ultrasonic bath.
2. Coupling Scheme (Amine Reactivity) 2.1 Buffer solution: Prepare the protein/antibody in a buffer solution without primary amines (such as PBS, carbonate buffer solution), with a pH value of 8.3 - 8.5.
2.2 Reaction: Add the PA-JF646-NHS stock solution to the protein solution (usually the dye is present in a 5-10 times molar excess).
2.3 Cultivation: Incubate at room temperature or at 37°C for 1 hour.
2.4 Purification: Use gel filtration (rotating column) or dialysis to separate the coupled protein from the free dye.
3. Cell Labeling and Imaging 3.1 Application: Suitable for both living cells and fixed cells with PALM (Photo-Activated Localization Microscopy).
3.2 Activation: Activated by 365 nm light.
3.3 Imaging: The maximum excitation wavelength is approximately 650 nm; the maximum emission wavelength is approximately 664 nm.
3.4 Washing: If necessary, wash the cells with 1x PBS and 2% BSA three times before imaging.
3.5 Wash-Free Option: This dye is fluorescently active, enabling "wash-free" imaging because the unbound ligands remain in the dark state until they bind.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. .