AcidiFluor™ Series

AcidiFluor™ ORANGE Labeling Kit

[Ready-to-use labeling kit]

[Acidic pH detecting probe]

570-590 nm:Orange

AcidiFluor ORANGE-NHS (succinimidyl ester) is pH probe “AcidiFluor ORANGE” which bonds amino groups of protein etc., and not need condensing agent. Not only antibody or protein but also oligonucleotide which terminal amination can be labeled by NHS. The contents of this kit cover from labeling to purification. A portion of AcidiFluor ORANGE-NHS is included most appropriate amount for labeling 100 μg of IgG.

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC304 AcidiFluor™ ORANGE Labeling Kit 5 times labeling

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    General Information for AcidiFluor ORANGE Labeling Kit

    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.

    Kit contains:

    • AcidiFluor ORANGE-NHS× 5
    • Reaction Buffer 1.5 mL × 1
    • Washing Buffer 10 mL × 1
    • Mictrocentrifugal filter × 5

     

    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

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    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.

FAQ

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

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

  • Q Can I use for fixed samples?
    A

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

Reference

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