Updated: February 7, 2020

For research use only

AcidiFluor™ ORANGE-NHS
Table 1. Product information
Catalog no.
GC302
GC303

Product nameAcidiFluor
ORANGE-NHSAmount1 mg
5 μg × 5

Storage upon receipt

Stability≤−20°C, keep desiccated1 year (when unopened

and protected from light.

and stored as described.)

1. About AcidiFluor ORANGE-NHS
AcidiFluor ORANGE-NHS is an acidic pH detecting probe

monitor pH changes along time, because of its reversibility

which is used for protein labeling via N-hydroxysuccin-of the fluorescence intensity upon pH changes, lowimide ester, just by mixing. Its fluorescent intensity

cytotoxicity and the slow photobleaching.

increases ~20-fold at an acidic pH (Exmax 544 nm, Emmax

By using 1 mg vial of GC302, you could label 10-50 mg

565 nm, ε = 80,000 M-1cm-1, QY. = 0.7 at pH 3.0),

antibody protein, whereas by using 1 vial of GC303 (5 μg),

compared to that in a physiological pH. It can be used to

you could label ~100 µg of antibody protein.

Absorption / fluorescence spectra of AcidiFluor ORANGE-NHS in each pH, and pH responsibilityof BSA-conjugated reagent (λex 532 nm/λem 568 nm.).

■ Storage
This product is shipped in a nitrogen gas-filled vial. Upon receipt, store the product desiccated and protected from light at≤−20°C. Dissolve the solid just before use. Before opening the vial, spin down the solid to the bottom and warm the vial to
the room temperature.

2.

An example protocol for labelingantibody■ Materials required but not provided
・

0.1 M sodium bicarbonate (NaHCO3)

・Anhydrous DMSO

・Ultrafiltration column, gel filtration or dialysis tube

4. Remove unreacted dye by using gel-filtration column
by exchanging buffer solution with PBS (pH 7.4).

■ Calculating degree of labeling
The degree of labeling (DOL) by AcidiFluor ORANGENHS can be calculated by the following equation:/

DOL =with a suitable molecular size.

,.
/■ Preparation of reagent and fluorescencelabeling/,

Where the parameters represent as:
A551, A280 : Absorbance of AcidiFluor ORANGE-conjugatedprotein at 551 nm and 280 nm, respectively

1. Dissolve the protein you wish to label in the 0.1 M

CF280/551 : A correction factor for the absorbance (see Table

NaHCO3 buffer to 2-10 mg/mL. Use gel-filtration

2.)

column to exchange the buffer if the protein is already,dissolved in a neutral buffer solution. Refer themanufacture’s instruction for the gel-filtration column.
Note: Amine-containing reagents such as BSA, other.

: An extinction coefficient of AcidiFluor ORANGENHS at 551 nm in pH 7.4 (see Table 2.):An extinction coefficient of a protein at 280 nm. For
IgG, 210,000 M-1cm-1.

protein, Tris, or glutathione inhibit the labelingreaction. In these cases, purify the protein before thelabeling to remove the extra amines.

Table 2. Properties of AcidiFluor ORANGE-NHS in pH 7.4

λAbs (nm)ε280,pH 7.4

CF280/551

551

62,3000.24

2. For GC302 user, add 107 μL of DMSO to the 1 mg vialof AcidiFluor ORANGE-NHS, mix well to prepare 10mM solution. Add this solution to the protein solution

Note: The properties are for quantification of theprepared in the previous step, at the molar ratio of 2 todye in pH 7.4 PBS. These are different in acidic

5. Immediately mix the solution by pipetting. Forenvironment. At pH 3.0, it shows maximum

GC303, directly add the protein solution to the vial, andabsorption ε = 80,000 M-1cm-1 at 544 nm.

immediately mix the solution by pipetting to dissolve
the AcidiFluor ORANGE-NHS solid.

■ Fluorescence detection

3. Incubate the mixture at 25 oC for 60 minutes. Protect

For laser excitation, either 532 nm or 514 nm is appropriate.

from light during the incubation. Mix the solution byFluorescence around 565 nm will be detected. For

tapping the tube in every 15 minutes.

fluorescent microscopy, general green excitation filter set
such as that for Cy3 can be used.

Table 3. Related Products
Catalog no.

Product name

Major applications

GC301

AcidiFluor ORANGE

GC304

AcidiFluor ORANGE Labeling Kit

All-in-one package for antibody labeling.

GC305

AcidiFluor ORANGE-Zymosan A

For the detection of phagocytosis.

GC306

AcidiFluor ORANGE-Dextran 10k

For the detection of endocytosis.

GC309

AcidiFluor ORANGE-Transferrin

For the detection of endocytosis.

GC310-01

HaloTag® AcidiFluor ORANGE ligand

For imaging of lysosome

Acidic pH indicator conjugated with HaloTag® ligand.

GORYO Chemical, Inc.
EAREE Bldg. 5F, Kita 8 Nishi 18-35-100, Chuo-ku, Sapporo 060-0008, JAPANTEL: +81-11-624-5860
https://goryochemical.com/