HaloTag® AcidiFluor™ORANGE Ligand



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Code No.ProductSizePrice
GC310-01HaloTag® AcidiFluor™
30 nmol$ 780.00
GC310-0260 nmol$ 1,500.00
  • HaloTag® ligand of acidic pH indicating fluorescent probe, AcidiFluor™ ORANGE.
  • High photostability enabling time-lapse imaging for long periods.
Name Exmax (nm)FLmax (nm) pKaε (M-1 cm-1) *1Φ *2
HaloTag® AcidiFluor™
5205655.060,0000.7 *3

*1.  Molar extinction coefficient
*2.  Quantum efficiency
*3.  It changes depending on the solution pH.

HaloTag® AcidiFluor™ ORANGE Ligand is a fluorescent probe which reversibly fluoresces in acidic pH. This probe shares the same fluorescent scaffold with AcidiFluor™ ORANGE, or RhP-EF and has HaloTag® ligand which covalently binds HaloTag® protein The fluorescence intensity of the Halotag® AcidiFluor™ reversibly increases in acidic pH.

Figure 1. (Left) Fluorescent spectra in pH 5.0 and7.5.
(Right) Fluorescence intensity in various pH solutions.

An example application

Figure 2.  Time lapse imaging of endocytosis and exocytosis using HaloTag® AcidiFluor™ ORANGE Ligand. HaloTag® AcidiFluor™ ORANGE Ligand is bound to EGFR via HaloTag®  which is exposed to the cell surface. It does not show fluorescence on the cell surface because it is exposed to the extracellular neutral buffer solution. Acidification of endocytotic vesicles comprising EGFR increases the fluorescence due to the lower pH. On the other hand, exocytosis of the vesicle instantaneously increases the pH and reduces the fluorescence significantly

(Method) A vector carrying HaloTag®-EGFP was transfected to RBL-2H3 cells using ScreenFect™ A (Wako). 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.


Masayuki Isa, Daisuke Asanuma, Shigeyuki Namiki, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, and Kenzo Hirose
“High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.” ACS Chem Biol 2014; 9(10) :2237-2241.

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