BODIPY-FL NHS ester

BODIPY FL NHS ester (BODIPY FL, SE) is a cell membranes-penatrable amine-reactive fluorescent probe. The maximum excitation/emission wavelength of the BODIPY-FL NHS ester are 502/511 nm, respectively. BODIPY-FL NHS ester has high stability and is insensitive to the polarity, pH and type of solvent, and can maintain stable fluorescence properties under different environmental conditions. BODIPY-FL NHS ester can be used for the synthesis of protease substrates, live cell imaging, protein labeling and immunoassay^[1][2]. In Vitro: 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 (Example for BODIPY-FL NHS ester)
Add anhydrous DMSO into the vial of BODIPY-FL NHS ester to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of BODIPY-FL NHS ester required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of BODIPY-FL NHS ester to protein is about 10.
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL BODIPY-FL NHS ester 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.