Thermo Fisher Scientific pHrodo User Manual
USER GUIDE
pHrodo™ Deep Red TFP Ester
Catalog Numbers P35358 and P35359
Pub. No. MAN0019655 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The pHrodo™ Deep Red TFP Ester, amine reactive dye readily reacts with a biomolecule’s primary amines to yield a covalently attached
fluorogenic pH probe. pHrodo Deep Red TFP ester enables you to label primary amines on a protein, cell, or virus to create a stable
conjugate that only fluoresces in acidic environments. Our low background pHrodo Deep Red makes it easier than ever to study
internalization with higher certainty of results and less optimization as pHrodo Deep Red only turns on in the late endosome and
lysosome. The pHrodo Deep Red dye has an excitation and emission maxima of approximately 640 nm and 655 nm respectively and can
be detected with standard Cy5 filters.
pHrodo Deep Red is a low-background pH sensor dye that shows no signal in neutral conditions and only fluoresces in acidic
environments. This unique property enables rapid assay development and certainty of results investigating antibody internalization,
endocytic and phagocytic pathways. pHrodo Deep Red enables better discrimination of internalized cargo from outside the cell because
it has an approximate pKa of 5 and will not turn on until it enters the late endosome and lysosome. pHrodo Deep Red dye can be
detected using a Cy5 fluorescent filter set and has been verified for use in a variety of applications, including flow cytometry, fluorescent
microscopy, and high content screening (HCS).
Here, we describe a quick and general protocol for using the amine-reactive form of the pHrodo™ Deep Red dye to label purified proteins
or antibodies in solution.
Contents and storage
Contents
pHrodo™ Deep Red TFP Ester, amine reactive (MW~1300) P35358 3 x 100 μg
pHrodo™ Deep Red TFP Ester, amine reactive (MW~1300) P35359 1 mg
Approximate fluorescence excitation and emission maxima of pHrodo Deep Red: 640/655 nm
[1]
The product is stable for at least 6 months when stored as directed.
Required materials not supplied
Unless otherwise indicated, all materials are available through
thermofisher.com. "MLS" indicates that the material is available
from fisherscientific.com or another major laboratory supplier.
Item Source
1 M sodium bicarbonate, pH 8.5 MLS
(Optional) Gel filtration column or media with
a suitable molecular weight cuto, equilibrated
with the buer of your choice:
Zeba™ Dye and Biotin Removal Columns
(Optional) Pierce™ Coomassie Plus (Bradford)
Assay Kit
(Optional) 4.3 % (wt%) phosphoric acid for
DOL determination
A44296S or A44298
23236
MLS
Cat. No. Amount Storage
Guidelines for Labeling Reaction
• Do not prepare the pHrodo™ Deep Red reactive dye stock
solution until ready to start the labeling reactions. This
reactive dye hydrolyzes readily and therefore should be used
immediately.
Guidelines for protein preparation
The purified protein should be at a concentration of 2.2 mg/mL in
a buer that does not contain primary amines (e.g., ammonium
ions, Tris, glycine, ethanolamine, triethylamine, glutathione), or
imidazole.All of these substances significantly inhibit protein
labeling.
Partially purified protein samples or protein samples containing
carriers such as BSA (e.g., antibodies) will not be labeled well
and should be purified prior to labeling. The presence of low
concentrations (<0.1% (w/v) of biocides, including sodium azide
and thimerosal, will not significantly aect labeling reaction.
[1]
• ≤–20°C
• Dessicate
• Protect from light
For Research Use Only. Not for use in diagnostic procedures.
To aid in removing low molecular weight components from the
protein sample (desalting) prior to labeling, it is possible to use
dialysis or small scale gel filtration. For dialysis we recommend
using the Slide-A-Lyzer™ Dialysis Cassettes (thermofisher.com).
We recommend PBS pH 7.2-7.5 as a prelabeling dialysis buer,
although 100 mM sodium bicarbonate buer can also be used.
(Optional) Guidelines for determining the degree of labeling (DOL)
• Several spectrophotometric methods are available for
determining the DOL of pHrodo™ Deep Red dye–labeled
conjugates. They are based on obtaining the conjugates
concentration by absorbance at 280 nm (A
(A
).
640
• We recommend using a NanoDrop™ spectrophotometer
to analyze the labeled antibody spectrophotometrically.
NanoDrop™ instruments (available from thermofisher.com)
require only 1–2 μL of sample and are full-featured UV/Vis
instruments.
• Determination of DOL for the conjugates prepared using the
kit are accurate only when they are diluted using a 4.3 %
(wt %) phosphoric acid solution. We recommend diluting the
antibody conjugate samples 1:3 in 4.3 % (wt %) phosphoric
acid before measuring the absorbance.
Note: This procedure will likely destroy the conjugate sample
and will not make the sample recoverable.
• Excessive dilution of some antibodies with low intrinsic A
may prevent you from deriving accurate A
samples. Use only a portion of your antibody conjugate
sample and dilute it only to the minimum volume necessary
for your cuvettes and spectrophotometer to avoid readings
below the optimal range for your instrument.
) and 640 nm
280
values for your
280
280
Label antibodies with pHrodo Deep Red aminereactive dye
This protocol describes the labeling of 1 mg of whole IgG with a
single, 100 μg aliquot of amine-reactive pHrodo™ Deep Red dye.
Briefly, the antibody is prepared at 2.2 mg/mL in PBS or similar
buer free of primary amines, and the dye is prepared at ~0.38
mM (0.5 mg/mL) in water.
Antibody labeling reaction
1. Prepare 1 mg of the antibody at 2.2 mg/mL in PBS or a
similar neutral buer free of primary amines.
2. Add 50 μL of a 1 M sodium bicarbonate to the antibody
solution.
3. Dissolve the contents of the 100 μg vial of pHrodo™ Deep
Red amine-reactive dye in 100 μL of water to prepare a
~0.77 mM (1 mg/mL) labeling solution. Completely dissolve
the contents of the vial by pipetting up and down.
IMPORTANT!
and discard any leftover solution.
Prepare this solution immediately before use
5.
Incubate the reaction for 2 hours at room temperature,
protected from light.
6. If desired, purify the conjugated antibody using a spin
column such as Zeba™ Dye and Biotin Removal Columns
(A449296S, A44298).
7. Antibody is ready to use.
Label purified proteins with pHrodo Deep Red
amine-reactive dye
The pHrodo™ Deep Red amine-reactive dyes readily react with a
protein’s primary amines to yield a covalently attached fluorogenic
pH probe. Here, we describe a general protocol for using the
amine-reactive forms of the pHrodo™ dye to label purified
proteins or antibodies in solution.
General protein labeling reaction
1. Prepare 1 mg of the protein at 2.2 mg/mL in PBS or a similar
buer free of primary amines.
2. Add 50 μL of a 1 M sodium bicarbonate to the antibody
solution.
3. Dissolve the contents of the 100 μg vial of pHrodo™ Deep
Red amine-reactive dye in 100 μL of water to prepare a
~0.77 mM (1 mg/mL) labeling solution. Completely dissolve
the contents of the vial by pipetting up and down.
IMPORTANT!
and discard any leftover solution.
4. Based on the amount of protein you wish to label, determine
the amount of reactive dye to use that will give you a dye
to protein molar ratio (MR) of 5–20 moles of dye per mole of
protein.
For IgG we recommend a starting molar ratio of 10.
5. Add the appropriate amount of reactive dye to the protein
solution in sodium bicarbonate buer and mix by pipetting
up and down several times.
6. Incubate the reaction for 2 hours at room temperature,
protected from light.
7. If desired, purify the conjugated protein using a spin column
such as Zeba™ Dye and Biotin Removal Columns (Cat. No.
A449296S, A44298).
Prepare this solution immediately before use
Purify the protein conjugates
We recommend using Zeba™ Dye and Biotin Removal Columns
(A449296S, A44298) or equivalent gel filtration media with an
appropriate molecular weight cuto.
To purify your conjugate by column chromatography, after elution
add bovine serum albumin (BSA) or any other stabilizer of choice
to a final concentration of 1–10 mg/mL to prevent denaturation.
4. Add all 100 uL of the ~0.77 mM labeling solution from to the
antibody solution and mix by pipetting up and down several
times.
2 pHrodo
™
Deep Red TFP Ester User Guide
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