AcidiFluor™ Series HaloFluor™ Series

HaloTag® AcidiFluor™ ORANGE Ligand

[Acidic pH detecting probe combined with HaloTag]

570-590 nm:Orange

This reagent is an acidic pH indicator, AcidiFluor ORANGE fused with  HaloTag® ligand, which forms covalent bond with HaloTag. It shows quite weak fluorescence at neutral pH, but shows strong fluorescence at acidic pH. 

Since it is cell impermeable reagent, it an be used to label membrane-associated HaloTag fused proteins and monitor the repetitive endocytosis and exocytosis cycle of the target proteins.

HaloTag® is a registered trade mark of Promega Corporation.  Goryo Chemical, Inc. produces HaloTag products under the license of Promega Corporation.


Available through Merck KGaA (Darmstadt, Germany) as:
SCT212-30nmol  HaloTag® AcidiFluor™ORANGE Ligand
SCT212-60nmol  HaloTag® AcidiFluor™ORANGE Ligand



Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC310-01 HaloTag® AcidiFluor™ ORANGE Ligand 30 nmol SCT212-30nmol HaloTag® AcidiFluor ORANGE Ligand
GC310-02 HaloTag® AcidiFluor™ ORANGE Ligand 60 nmol SCT212-60nmol  HaloTag® AcidiFluor ORANGE Ligand


  • Protocol

  • Flyer

  • SDS

  • Product Information


    Properties of HaloTag AcidiFluor ORANGE ligand

    Product name
    target reaction pKa
    Absmax (nm) FLmax (nm) ε Φ*
    HaloTag AcidiFluor ORANGE ligand pH reversible 5.0 520 565 60,000 0.7

    *Maximum quantum yield. It changes upon pH.



    Left, fluorescence spectra at pH 5.0 and 7.5. Right, Fluorescence intensity change upon pH.


  • Imaging example with HaloTag® AcidiFluor™ ORANGE Ligand


    Imaging of endocytosis/exocytosis cycle using HaloTag® AcidiFluor™ ORANGE Ligand

    When HaloTag AcidiFluor ORANGE Ligand was applied to the cells which was exogeneously expressed HaloTag-EGFR,  fluorescence is not observed when the labeled receptors exposed to the neutral extracellular fluid. Upon endocytosis of the receptor-containing membrane and acidification of the vesicle, fluorescence increases.  Upon exocytosis of the vesicle, the pH instantaneously neutralizes by the extracellular fluid and fluorescence disappears.

    A vector carrying HaloTag®-EGFP was transfected to RBL-2H3 cells using ScreenFect™ A (Wako Pure Chemicals). After 6-hour culture, 1 μg/mL of anti-DNP IgG was added to the medium and continued culture for 16 hours. After exchanging the medium, 2 μM of HaloTag® AcidiFluor™ ORANGE Ligand in culture medium was reacted with the cells for 30 min at 37oC, then washed with HBSS 2 times and observed by microscopy. Endocytosis and exocytosis was induced by the addition of 50 μg/mL DNP-BSA (final conc.). Images were obtained using NIKON Ti-E, PlanFluor 40/1.3 and Hamamatsu ORCA-R2. Magenta color indicates the fluorescence of  AcidiFluor™ ORANGE. (Imaging was performed at NIKON Imaging Center,Hokkaido University)



  • 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 fluorescence intensity at pH less than 3.

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

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


N. Scheefhals, L. A. E. Catsburg, M. L. Westerveld, T. A. Blanpied, C. C. Hoogenraad, H. D. MacGillavry (2019)
Cell Rep. 29: 258-269.e8 DOI: 10.1016/j.celrep.2019.08.102

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