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RatioWorks™ BCFL, SE

The pH dependent absorbance spectra of BCFL Acid.
The pH dependent absorbance spectra of BCFL Acid.
The pH dependent absorbance spectra of BCFL Acid.
The pH dependent emission spectra of BCFL Acid.
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Physical properties
Molecular weight~700
Spectral properties
Excitation (nm)504
Emission (nm)527
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
Intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport, endocytosis, chemotaxis, cell adhesion and other cellular processes. pH-sensitive fluorescent dyes have been widely applied to monitor changes in intracellular pH in recent years. Imaging techniques that use fluorescent pH indicators also allow researchers to investigate these processes with much greater spatial resolution and sampling density that can be achieved using other technologies such as microelectrode. Among them, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is the most popular pH probe since it can be used to monitor cellular pH ratiometrically. However, all the commercial BCECF AM is a complex mixture of at least three isomers with different ratios from batch to batch, complicating the BCECF applications. BCFL is developed to overcome this isomer difficulty associated with BCECF AM. As BCECF, BCFL exhibits a pH-dependent dual excitation, essentially identical to BCECF. It has pKa of ~7.0, identical to BCECF too. As with BCECF, the dual excitation spectrum of BCFL with an isosbestic point at 454 nm should make BCFL a good excitation-ratiometrie pH indicator. BCFL ratiometric imaging makes intracellular pH determination essentially independent of several variable factors, including dye concentration, path length, cellular leakage and photobleaching rate. BCFL, AM is a single isomer, making the pH measurement much more reproducible than the BCECF, AM, which is consisted of quite a few different isomers. This amino-reactive BCFL derivative can be used for preparing BCFL conjugates as ratiometric fluorescent probes.

Example protocol


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

2. RatioWorks™ BCFL, SE stock solution (Solution B)
Add anhydrous DMSO into the vial of RatioWorks™ BCFL, SE to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.


This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with RatioWorks™ BCFL, SE. You might need further optimization for your particular proteins. Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction
  1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes. 

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. Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried. 


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Spectral properties

Excitation (nm)504
Emission (nm)527



View all 2 citations: Citation Explorer
The F0F1 ATP synthase regulates human neutrophil migration through cytoplasmic proton extrusion coupled with ATP generation
Authors: Gao, Jun and Zhang, Tian and Kang, Zhanfang and Ting, Weijen and Xu, Lingqing and Yin, Dazhong
Journal: Molecular Immunology (2017): 219--226
Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells
Authors: Wu, Yiqing and Zhang, Min and Liu, Rui and Zhao, Chunjie
Journal: Yonsei Medical Journal (2016): 1252--1259


View all 34 references: Citation Explorer
Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy
Authors: Alvarez-Leefmans FJ, Herrera-Perez JJ, Marquez MS, Blanco VM.
Journal: Biophys J (2006): 608
Photophysics of the fluorescent pH indicator BCECF
Authors: Boens N, Qin W, Basaric N, Orte A, Talavera EM, Alvarez-Pez JM.
Journal: J Phys Chem A Mol Spectrosc Kinet Environ Gen Theory (2006): 9334
Drug efflux transport properties of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (bcecf-am) and its fluorescent free acid, bcecf
Authors: Bachmeier CJ, Trickler WJ, Miller DW.
Journal: J Pharm Sci (2004): 932
A rapid method for measuring intracellular pH using BCECF-AM
Authors: Ozkan P, Mutharasan R.
Journal: Biochim Biophys Acta (2002): 143
Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate
Authors: Olson DP, Taylor BJ, Ivy SP.
Journal: Cytometry (2001): 105
In vivo application of intestinal ph measurement using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (bcecf) fluorescence imaging
Authors: Marechal X, Mordon S, Devoisselle JM, Begu S, Guery B, Neviere R, Buys B, Dhelin G, Lesage JC, Mathieu D, Chopin C.
Journal: Photochem Photobiol (1999): 813
Fluorescence probe (BCECF) loading efficiency in human platelets depends on cell concentration: application to pHi measurements
Authors: Ruiz-Palomo F, Garcia C, Gomez M, Revenga M.
Journal: Clin Biochem (1999): 391
Analysis of the uptake of the fluorescent marker 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (bcecf) by hydrogenosomes in trichomonas vaginalis
Authors: Scott DA, Docampo R, Benchimol M.
Journal: Eur J Cell Biol (1998): 139
Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF
Authors: Weinlich M, Heydasch U, Mooren F, Starlinger M.
Journal: Res Exp Med (Berl) (1998): 73
BCECF in single cultured cells: inhomogeneous distribution but homogeneous response
Authors: Weinlich M, Theiss C, Lin CT, Kinne RK.
Journal: J Exp Biol (1998): 57