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[pH probe for labeling protein and nucleic acid]AcidiFluor™ ORANGE-NHS

  • Just mixing
  • High S/N ratio
  • High responsivity after labeled to protein

AcidiFluor™ ORANGE-NHS is pH probe for labeling protein and nucleic acid. This probe is added the labeling parts for amino group with AcidiFluor™ ORANGE which is a fluorescence imaging probe which enhances fluorescence dramatically in acidic environments. All you have to do is mixing AcidiFluor™ ORANGE-NHS with amino group like antibody and protein and forming stable covalent bond through the intermediary of N-hydroxysuccinimidyl ester (NHS). This product can be used monitoring pH under local environment and detecting endocytosis / phagocytosis. Even AcidiFluor™ ORANGE-NHS after labeling with protein, its excellent selectivity enables to detect acidic environment. Namely, in the condition of pH5.0, identical to the environment of acidic organelles, fluorescence intensity is 10-fold or more compared to physical pH 7.4. AcidiFluor™ ORANGE shows great photostability against irradiating excitation light. As it emits orange color fluorescent by excitation at 544 nm, multicolor imaging is possible by combining with blue fluorescence (CFP, Hoechst, etc.), green fluorescence (GFP, fluorescein, etc.) and near-infrared fluorescence. Furthermore, not only microscope imaging but evaluating of uptake the compounds using fluorescence plate reader.

Fig 1. Reaction of AcidiFluor™ ORANGE-NHS and antibody.
Fig 1. Reaction of AcidiFluor™ ORANGE-NHS and antibody.

The feature of AcidiFluor™ ORANGE-NHS

λabs 544 nm λfl 565 nm pKa 5.3, 6.8

ε 80,000

High S/N ratio

Figure 2. Comparative Change in Fluorescent Intensity
Figure 2. Comparative Change in Fluorescent Intensity

Fluorescent intensity of AcidiFluor NHS as the BSA labeled form were compared with competitor probes in phosphate buffer pH 5.0 or pH 7.4, respectively. Intensity of AcidiFluor™ORANGE-NHS in pH5.0 increased about 20 fold compared to pH7.4, while other probes showed only 1.8 fold or 7.5 fold increase in fluorescent intensity. 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) : λex 560nm / /λem 582 nm
CypHer™5E-NHS (GE Healthcare) : λex 644 nm / /λem 667 nm

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

Fig 3. Fluorescence intensity time course showed graphically in cell body.

Fig 3. Fluorescence intensity time course showed graphically in cell body. AcidiFluor™ ORANGE-BSA was risen fluorescence intensiity with time when they were incorporated into a cell, but pHrodo-BSA and CypHer5E-BSA was not.

Reference
Hayashi A, Asanuma D, Kamiya M, Urano Y, Okabe S. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons Neuropharmacology Volume 100, January 2016, 66-75
Asanuma D, Takaoka Y, Namiki S, Takikawa K, Kamiya M, Nagano T, Urano Y & Hirose K. Acidic-pH-Activatable Fluorescence Probes for Visualizing Exocytosis Dynamics. Angew Chem Int Ed 2014, doi:10.1002/anie.201402030.
Masayuki Isa, Daisuke Asanuma, Shigeyuki Namiki, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, and Kenzo Hirose, ACS Chem Biol 2014 Oct 22;9(10):2237-41, “High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.”
Watanabe R, Soga N, Fujita D, Tabata KV, Yamauchi L, Kim SH, Asanuma D, Kamiya M, Urano Y, Suga H and Noji H. Nature Communications 2014 Jul24; 5, Article number: 4519 doi:10.1038/ncomms5519 “Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity”
price
Code no. Product Labeling part size Price Protocol MSDS
GC302 AcidiFluor ORANGETM-NHS NHS-ester 1 mg USD 798.00 Donwload 651KB Donwload 159KB
GC303 5 samples USD 498.00 Donwload 322KB

Contact Information

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