Janelia Fluor 646, SE (JF646, SE) is a red fluorescent dye can be used in cellular imaging^[1]. Janelia Fluor products are licensed under U.S. Pat. Nos. 9,933,417, 10,018,624 and 10,161,932 and other patents from Howard Hughes Medical Institute.

Excitation maximum=646 nm; emission maximum=664 nm^[1].
JF646 can serve as ligand for self-labeling tag (such as HaloTag)^[1].
Janelia Fluor 646, SE (JF646, SE) is modified by JF646. JF646, a red fluorescent dye, is photostable, membrane-permeable, has a high extinction coefficient^[2].
Protocol
1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1 M sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
2.Dye Preparation
Add anhydrous DMSO into the vial of Janelia Fluor® 646, SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of Janelia Fluor® 646, SE required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of Janelia Fluor® 646, SE to protein is about 10.
Example: assuming the required marker protein is 500 μL 2 mg/mL IgG (MW=150,000), use 100 μL DMSO dissolve 1 mg Janelia Fluor® 646, SE, the required Janelia Fluor® 646, SE volume is 3.98 μL, and the detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG)/MW (IgG) =2 mg/mL × 0.5 mL/150,000 mg/mmol= 6.7×10^-6 mmol
2) mmol (Janelia Fluor® 646, SE) = mmol (IgG) × 10 = 6.7×10^-6 mmol×10 = 6.7 × 10^-5 mmol
3) μL (Janelia Fluor® 646, SE) = mmol (Janelia Fluor® 646, SE) ×MW (Janelia Fluor® 646, SE)/mg/μL (Janelia Fluor® 646, SE) = 6.7 ×10^-5 mmol ×593.70 mg/mmol / 0.01 mg/μL = 3.98 μL (Janelia Fluor® 646, SE)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL Janelia Fluor® 646, SE is slowly added to 0.5 mL protein sample
In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don'tmix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a SepHadex G-25 column.
1) Prepare SepHadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From "Run conjugation reaction") to the top of the SepHadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

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

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Grimm JB, et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods. 2015;12(3):244-250. doi:10.1038/nmeth.3256  [Content Brief]

  [2]. Basu S, et al. FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells. Nat Commun. 2018;9(1):2520. Published 2018 Jun 28.  [Content Brief]