AcidiFluor™ Series

AcidiFluor™ ORANGE-NHS

[Acidic pH detecting probe]

570-590 nm:Orange

AcidiFluor ORANGE-NHS is a chemical probe which reversibly fluoresces upon acidic pH, modified with N-hydroxysuccimide ester (NHS) which forms covalent bond with primary amines just by mixing. It can be used for labeling proteins, antibodies and other molecules.

For example, it can be applied for visualizing endocytosis of target protein on cell membrane by labeling antibodies that binds to the target protein.


Available through Merck KGaA (Darmstadt, Germany) as:
SCT214  BioTracker Orange-NHS Live Cell pH Dye



Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC302 AcidiFluor™ ORANGE-NHS 1 mg SCT214 BioTracker Orange-NHS Live Cell pH Dye
GC303 AcidiFluor™ ORANGE-NHS 5 μg × 5


  • Protocol

  • Flyer

  • SDS

  • Product Information


    General Information

    AcidiFluor ORANGE-NHS binds to primary amines just by mixing. Thus it can be used to label antibodies and other proteins which have lysine residues, as shown in the figure below.


    Properties of AcidiFluor ORANGE-NHS

    Product Name target reaction pKa
    Absmax (nm) FLmax (nm) ε Φ
    AcidiFluor ORANGE-NHS pH reversible 5.3, 6.8 544 565 80,000 0.7


    High S/N ratio

    Fluorescent intensities of three pH probes and their BSA labeled form were measured in phosphate buffer of pH 5.0 and that of pH 7.4. Intensity of AcidiFluor ORANGE-NHS shows ~20 times increase at pH 5.0 compared to that at pH 7.4, while other probes showed only 1.8 times and 7.5 times increase in the fluorescence intensities, respectively. Only AcidiFluor ORANGE-NHS has high pH responsivity and dynamic range as both the NHS and BSA labeled forms.

    AcidiFluor™ ORANGE-NHS : λex 532 nm / λem 568 nm
    pHrodo™ Red-NHS (Life Technology社) : λex 560nm / /λem 582 nm
    CypHer™ 5E-NHS (GE Healthcare社) : λex 644 nm / /λem 667 nm


    A timecourse of fluorescence intensity changes during endocysosis, measured by choosing a spot on a cell. Fluorescence increase of AcidiFluor ORANGE-BSA was most significant compared to BSA labeled with other pH probes.


    Observation of AcidiFluor™ ORANGE – BSA endocytosis (HeLa cells).

  • Cell imaging example using AcidiFluor ORANGE-NHS and AcidiFluor-ORANGE Zymosan A


    Cell imaging example using AcidiFluor ORANGE-NHS and AcidiFluor-ORANGE Zymosan A

    Uptake of AcidiFluor ORANGE labeled anti-EGFR antibody by A431 cell (EGFR overexpressing cell line)

    Anti-EGFR antibody was labeled with AcidiFluor ORANGE-NHS, and unreacted dye was removed by ultrafiltration. A431 cell, EGFR overexpressing cell line, was treated with purified AcidiFluor ORANGE labeled anti-EGFR antibody, then time-lapse observation was performed by using confocal microscope. After 120 min of the addition, fluorescence signal derived from AcidiFluor ORANGE began to be detected, and further 60 min later, strong fluorescence was observed. This result indicates that AcidiFluor ORANGE labeled anti-EGFR antibody was incorporated into the cells by endocytosis and then the endocytotic vesicle was acidified.


    Phagocytosis of AcidiFluor ORANAGE-Zymosan A by RAW264.7 cells

    After addition of AcidiFluor ORANGE-Zymosan A to RAW264.7 cells, the increase of fluorescence intensity was observed, indicating an acidification of folliculi.


  • Q Tell me the fluorescence intensity at pH less than 3.

    Intensity of AcidiFluor ORANGE is almost constant at the pH 3 or less.

  • Q Can I use for fixed samples?

    No, basically, it cannot be applied to fixed cells. Acidic pH of lysosomes and endosomes are kept by the activity of living cells and fixed (dead) cells do not keep the acidic pH.

    On the other hand it could be possible to detect localization of the probes by soaking fixed cells into an acidic buffer, because localization of AcidiFluor ORANGE, which has a structure to be localized in lysosomes, antibody-labeled AcidiFuor ORANGE-NHS and HaloTag labeled HaloTag AcidiFluor ORANGE ligand can be kept for a while after fixation.


  • Q Tell me the efficient labeling method using NHS reagent.

    Prepare purified antibody (or other proteins). If you intended to crude protein sample, we recommend to purify protein using either affinity column, ultrafilteration or gel filteration before the labeling to increase labeling efficiency. In addition, avoid to use Tris-buffer, because Tris has primary amine and it strongly inhibit the reaction of NHS to the target protein.

    In some cases, reaction at 4oC for overnight gives higher degree of labeling compared with reaction at 37oC for 1 hour.

  • Q How can I get molar extinction coefficient of a protein at 280 nm?

    Molar extinction coefficient of protein at 280 nm can be calculated by the methods of Gill and von Hippel (1989) Analytical Biochemistry, 182: 319-326  and Anthis and Clore (2013) Protein Science 22:851-858.

    You may find Web services to obtain them in the following sites. Goryo Chemical do not support the usage of these sites and use them according to the Terms and Conditions in each sites.

  • Q How can I prepare 0.1 M sodium bicarbonate buffer? Why this buffer is required?

    Dissolve sodium bicarbonate (NaHCO3) to pure water and adjust the concentration to be 0.1 M. The pH should be in the range of 8.0 to 8.4. Alternatively, you can adjust pH by adding a small amount of Na2CO3  or  HCl.

    The reason to recommend this buffer is that the reaction of NHS ester and primary amines is more efficient at alkaline pH. Instead, you can use other alkaline buffers such as HEPES/phosphate/borate buffers.  Avoid to use Tris buffer because it has primary amines and inhibit the reaction of NHS with the target molecules.

  • Q My question is not in this FAQ list.....


K. Kawakatsu, M. Maemura, Y. Seta, T. Nishikata (2021)
Anticancer Res. 41: 4089-4092 DOI:10.21873/anticanres.15211

J. Ogata, K. Hirao, K. Nishioka, A. Hayashida, Y. Li, H. Yoshino, S. Shimizu, N. Hattori, Y. Imai (2021)
Int. J. Mol. Sci. 22: 3708. DOI: 10.3390/ijms22073708

T. Nakatani, K. Tsujimoto, J. H. Park, T. Jo, T. Kimura, Y. Hayama, H. Konaka, T. Morita, Y. Kato, M. Nishide, S. Koyama, S. Nada, M. Okada, H. Takamatsu, A. Kumanogoh (2021)
Nat. Commun. 12:3333 DOI: 10.1038/s41467-021-23654-3

M. Ishikawa, R. Mashiba, K. Kawakatsu, N. K. Tran, T. Nishikata (2018)
Macrophage 5: e1627  DOI: 10.14800/Macrophage.1627

R. Mashiba, M. Ishikawa, Y. Sumiya, K. Kawakatsu, N. K. Tran, T. Nishikata (2018)
Anticancer Res. 38: 4295-4298 DOI:10.21873/anticanres.12727

A. Hayashi, D. Asanuma, M. Kamiya, Y. Urano, S. Okabe (2016)
Neuropharmacology 100: 66-75 DOI:10.1016/j.neuropharm.2015.07.026

D. Asanuma, Y. Takaoka, S. Namiki, K. Takikawa, M. Kamiya, T. Nagano, Y. Urano, K. Hirose (2014)
Angew. Chem. Int. Ed. Engl. 53: 6085-6089 DOI:10.1002/anie.201402030

M. Isa, D. Asanuma, S. Namiki, K. Kumagai, H. Kojima, T. Okabe, T. Nagano, K. Hirose (2014)
ACS. Chem. Biol. 9: 2237-2241 DOI:10.1021/cb500654q

R. Watanabe, N. Soga, D. Fujita, K. V. Tabata, L. Yamauchi, S. H. Kim, D. Asanuma, M. Kamiya, Y. Urano, H. Suga, H. Noji (2014)
Nat. Commun. 5, Article number: 4519 DOI:10.1038/ncomms5519