Thermo Fisher Scientific Protein Labeling Kits User Manual
USER GUIDE
Protein Labeling Kits (For Alexa Fluor™, Pacific Blue™, Fluorescein-EX,
and Oregon Green™ 488)
Pub. No. MAN0019835 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 Protein Labeling Kits provide a convenient means to covalently label proteins with the superior Alexa Fluor™, Pacific Blue™, Fluorescein-EX,
and Oregon Green™ 488 fluorescent dyes. These kits are optimized for labeling 1 mg of IgG per reaction and comparable amounts of other
proteins (>20 kDa) can also be labeled. For labeling smaller amounts of antibodies (~100 µg), we recommend our antibody labeling kits.
Each Protein Labeling Kit contains everything you need to perform 3 separate labeling reactions and purify the resulting conjugates. The reactive
dye has either a succinimidyl ester (SE) or a tetrafluorophenyl (TFP) ester moiety that reacts eciently with primary amines of proteins to form
stable dye–protein conjugates. Each of the 3 vials of reactive dye provided in the kit is sucient for labeling ~1 mg of an IgG antibody, although
other proteins can also be labeled.
Alexa Fluor™ dyes produce protein conjugates that are typically brighter with better photostability and lower pH sensitivity relative to the traditional
dyes (e.g., AMCA, fluorescein/FITC, TRITC, etc.) and the Cy™ dyes (Table 1). Unlike other dyes, the fluorescence of Alexa Fluor™ conjugates are
insensitive to pH between pH 4 and 10. Also, various Alexa Fluor™ dyes are resistant to quenching at high degrees of substitution and thus are
brighter than similar proteins labeled with other dyes. Alexa Fluor™-labeled antibodies can be used for multiple applications, including fluorescent
microscopy, flow cytometry, western blotting, ELISA, and indirect FISH. Pacific Blue™ is a unique dye designed for use with a violet laser (405 nm)
for flow cytometry applications. Dye spectra can be found at thermofisher.com
Table 1 Properties of Alexa Fluor™, Pacific Blue™, Fluorescein-EX, and Oregon Green™ 488 dyes.
Commonly
Cat. No.
A10170 Alexa Fluor™ 350 346/442 Blue
A10235 Alexa Fluor™ 488 494/519 Green
A10236 Alexa Fluor™ 532 530/554 Yellow
A10237 Alexa Fluor™ 546 554/570 Orange
A20174 Alexa Fluor™ 555 555/565 Orange
A10238 Alexa Fluor™ 568 577/603 Orange/Red
A10239 Alexa Fluor™ 594 590/617 Red
A20170 Alexa Fluor™ 633 632/647 Far Red
A20173 Alexa Fluor™ 647 650/668 Far Red
A20171 Alexa Fluor™ 660 663/690 Far Red
A20172 Alexa Fluor™ 680 679/702 Infrared
F10240 Fluorescein-EX 494/518 Green
O10241 Oregon Green™ 488 496/524 Green
P30012 Pacific Blue
[1]
The fluorophores listed have similar excitation and emission properties but may vary in brightness, photostability, water solubility, quantum yield, and pH response relative to the
Alexa Fluor™, Pacific Blue™, Fluorescein-X, and Oregon Green™ 488 dyes.
[2]
Human vision is insensitive to light beyond ~650 nm, and, therefore, it is not possible to view the far-red–fluorescent dyes by looking through the eyepiece of a conventional
fluorescence microscope.
Dye Ex/Em Maxima (nm) Fluorescent Color Spectrally Comparable Fluorophores
AMCA, eBFP, DAPI, Hoechst 33342, Hoechst
Fluorescein/FITC, BODIPY™ FL, DiO, Cy2™,
Rhodamine 6G, BODIPY™ R6G, Qdot™ 545,
Tetramethylrhodamine/TRITC, Dil, Cy3™,
Qdot™ 565, mOrange/OFP
Tetramethylrhodamine/TRITC, R-PE,
BODIPY™ TMR, Cy3™, Qdot™ 565,
Rhodamine Red, Qdot™ 605, Cy3.5™, RFP,
Texas Red™, BODIPY™ TR, Qdot™ 625, HcRed
[2]
[2]
[2]
™
410/455 Blue Hoechst 34580, TagBFP
APC, Qdot™ 655, BODIPY™ 630/650-X Cy5
APC, Qdot™ 655, DDAO, DiD, DRAQ5™, TO-
PRO™-3, Cy5™, IRDye™ 650
Qdot™ 705, Cy5.5™, iRFP, IRDye™ 680,
Fluorescein/FITC, BODIPY™ FL DiO, Cy2™,
Fluorescein/FITC, BODIPY™ FL DiO, Cy2™,
33258
Qdot™ 525, GFP
mBanana
mOrange/OFP
DsRed
Tandem, mRaspberry
™
Cy5.5
IRDye™ 700
Qdot™ 525, GFP
Qdot™ 525, GFP
Note: DiO, DiI, DiD, and DiR are the lipophilic, carbocyanine dyes.
[1]
Used Filter
Texas Red
Cy5™, APC
Cy5.5™, LICOR
700 channel
Violet laser/DAPI
Sets
DAPI
FITC
R6G
TRITC
TRITC
RITC
™
Cy5.5
FITC
FITC
channel
™
™
For Research Use Only. Not for use in diagnostic procedures.
Contents and storage
Material Amount Storage
Reactive Dye
(Component A)
Sodium bicarbonate
(MW=84) (Component B)
Purification columns
(Component C)
Collection tubes 6 tubes
Number of labelings: Each vial of reactive dye contains the appropriate
amount of dye to label approximately 1 mg of IgG (MW ~145,000) as 0.5 mL
of IgG solution at 2 mg/mL.
[1]
The kit can be stored under the conditions listed. For optimal storage conditions of
individual components, refer to the labels on the vials or bags. Note that the reactive
dye (Component A) may be stored frozen at ≤−20°C or at 2–8°C. Do not freeze the
purification columns (Component C).
[2]
The resin in each column is supplied in a 0.1 M NaCl/0.05% sodium azide solution.
[2]
3 vials (each
containing a
magnetic stir bar)
84 mg
3 each
• Store at 2–
• Do not
[1]
6°C
protected
from light.
freeze.
Stability
When stored
properly, kit
components
are stable
for at least 3
months.
Equipment required but not supplied
• Benchtop centrifuge capable of 1,000 × g
Labeling protocol
Prepare the proteins
• For optimal labeling eciency, the purified protein must be in a
buer free of ammonium ions or primary amines.
• If the protein is in an unsuitable buer (e.g., Tris or glycine), the
buer should be replaced with phosphate-buered saline (PBS)
by dialysis or another method. Impure proteins (e.g., antibodies in
crude serum) will not label well.
• The presence of low concentrations of sodium azide (≤3 mM) or
thimerosal (≤1 mM) will not interfere with the conjugation reaction.
• This kit can be used to label virtually any protein, although the
following protocol has been optimized for labeling IgG antibodies.
Each vial of reactive dye contains the appropriate amount of dye
to label approximately 1 mg of IgG (MW ~145,000) as 0.5 mL of
IgG solution at 2 mg/mL.
For tips on optimizing the procedure for other proteins or for antibody
solutions at lower concentrations, see “Optimize the kit for use with
other proteins and/or concentrations” on page 4 or “Optimization
and troubleshooting” on page 4.
Label the protein
1. Prepare a 1 M solution of sodium bicarbonate by adding 1
mL of deionized water (dH2O) to the provided vial of sodium
bicarbonate (Component B). Vortex or pipet up and down until
fully dissolved. The bicarbonate solution, which will have a pH
8-9, can be stored at 4°C for up to 2 weeks.
2. If the protein concentration is greater than 2 mg/mL, the protein
should be diluted to 2 mg/mL in a suitable buer (e.g., PBS or
0.1 M sodium bicarbonate.
3. To 0.5 mL of the 2 mg/mL protein solution, add 50 µL of 1 M
bicarbonate prepared in step 1.
Note: Bicarbonate, pH~8.3, is added to raise the pH of the
reaction mixture, since TFP and succinimidyl esters react
eciently at alkaline pH.
4. Allow a vial of reactive dye to warm to room temperature.
Transfer the protein solution from step 3 to the vial of reactive
dye. This vial contains a magnetic stir bar. Cap the vial and invert
a few times to fully dissolve the dye. Stir the reaction mixture for
1 hour at room temperature.
Purify the labeled proteins
Thermo Scientific™ Zeba™ Dye and Biotin Removal Spin Columns in
this kit contain a ready-to-use resin that is uniquely designed for rapid
removal of non-conjugated fluorescent dyes with exceptional protein
recovery. The purification resin is designed to separate free dye from
proteins with MW >20 kDa. For smaller proteins, gel filtration media
of a suitable molecular weight cuto should be selected. Labeled
peptides may be separated from free dye by TLC or HPLC. Removal
of free dye after a labeling reaction is essential for the accurate
determination of dye to protein ratios. For optimal protein recovery
and dye removal, ensure that the appropriate amount of sample and
buer conditions are used.
IMPORTANT! Protein conjugates that are between 20-50 kDa require
a more alkaline buer system to elute and will retain on the column if
the buer system is not changed. See procedure below for purifying
20-50 kDa conjugates.
Procedural guidelines
• Do not reuse the purification resin.
• Limit DMF and other organic solvents to ≤10% of solvent volume
loaded onto the column.
• If labeling a 20-50 kDa protein, refer to “Purify 20-50 kDa
conjugates” on page 2 to ensure conjugate recovery.
Prepare the spin column
1. Twist to remove the bottom plug of the column, then loosen the
cap. Do not remove the cap.
2. Place the column in a collection tube, then centrifuge the
column-tube assembly at 1,000 × g for 2 minutes to remove the
storage buer. Discard the flowthrough.
3. If using a fixed angle rotor, place a mark on the side of the
column facing away from the rotor center. For all subsequent
centrifugation steps, place the column in the centrifuge with the
mark facing away from the rotor center.
IMPORTANT! Improper orientation of the column during
centrifugation can result in reduced small molecule removal.
4. If desired, the resin storage buer can be exchanged using a
buer of choice. To exchange, add 2 mL of equilibration buer to
the column, then centrifuge at 1,000 × g for 2 minutes. Discard
the flowthrough.
Purify 20-50 kDa conjugates
If purifying a 20–50 kDa protein, a buer exchange is required to
ensure conjugate recovery.
1. Following storage buer removal, apply 500 µL of 0.2 M, pH 9.4
bicarbonate buer to the column (Cat. No. 28382).
2. Centrifuge the column-tube assembly at 1,000 × g for 2 minutes.
3. For optimal conjugate recovery, repeat steps 1 and 2 two more
times for a total of 3 column washes to ensure equilibration.
Process the sample
1. Place the prepared column into a new collection tube, then
remove the cap.
2. Slowly apply the reaction mixture (~0.5 mL) to the center of the
settled resin.
3. Centrifuge the column-tube assembly at 1,000 × g for 2 minutes
to collect the sample. The sample will be in the collection tube
and the column can now be discarded.
4. (Optional) The column may be washed with an additional ~0.5
mL of suitable buer (e.g., PBS) to maximize the recovered
sample, applied as in steps 2 and 3. Note that this extra wash
step will dilute the recovered conjugate and may be omitted if
higher concentration is desired.
Determine the Degree of Labeling (Optional)
1. Dilute a small amount of the purified conjugate into PBS or other
suitable buer and measure the absorbance in a cuvette with a
1-cm path length at 280 nm (A
(λ
) for the respective dye (A
max
cuvette that may provide a shorter or longer path length, see
“Note B” on page 4 to modify the calculation.
) and the absorbance maximum
280
). If using a NanoDrop™ or
dye
2
Protein Labeling Kits User Guide (For Alexa Fluor™, Pacific Blue™, Fluorescein-EX, and Oregon Green™ 488)
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