USER GUIDE
Texas Red™-X Protein Labeling Kit
Catalog Number T10244
Pub. No. MAN0019833 Rev. A.0
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Product description
The Texas Red™-X Protein Labeling Kit provides a convenient means to label proteins with Texas Red™-X dye. This kit contains everything
that is required to perform 3 separate labeling reactions and purify the resulting conjugates. Each of the 3 vials of the reactive dye
provided in the kit is sucient for labeling ~1 mg of an IgG antibody, although other proteins can also be labeled.
The Texas Red™-X dye has a succinimidyl ester moiety that reacts eciently with primary amines of proteins to form stable dye–protein
conjugates. The succinimidyl ester group is separated from the fluorophore by a seven-atom spacer (X) to minimize interaction between
the fluorophore and the protein to which the dye is conjugated. Texas Red™-X dye–labeled proteins have absorption and fluorescence
emission maxima of approximately 595 nm and 615 nm, respectively.
Contents and storage
Material Amount Storage
Texas Red™-X Reactive Dye (Component A) 3 vials (each containing a magnetic
Sodium bicarbonate (MW=84) (Component B) 84 mg
Purification columns (Component C)
Dimethylsulfoxide (DMSO) (Component D) 1 × 200 µL
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 N NaCl/0.05% sodium azide solution.
[2]
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 or proteins stabilized with bovine
serum albumin (BSA) or gelatin) 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.
stir bar)
3 each
• Store at 2–6°C
protected from light.
• Do not freeze.
• 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 3 or
“Optimization and troubleshooting” on page 3.
[1]
When stored properly, kit
components are stable
for at least 3 months.
Stability
For Research Use Only. Not for use in diagnostic procedures.
Labeling reaction
Prepare the spin column
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 ~8.3, 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 succinimidyl esters react eciently at
alkaline pH.
4. Allow a vial of reactive dye to warm to room temperature.
Add 10 µL of DMSO (Component D) to the reactive dye
underneath the stir bar. Rotate the vial to allow the DMSO to
moisten and dissolve the reactive dye.
Note: The addition of DMSO helps to dissolve the
hydrophobic Texas Red™-X reactive dye into the aqueous
protein solution, thereby increasing the eciency of the
reaction.
5. Transfer the protein solution from step 3 to the vial of
reactive dye with the added DMSO. Cap the vial and invert a
few times to fully dissolve the dye. Stir the reaction mixture
for 1 hour at room temperature.
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 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.
If desired, the resin storage buer can be exchanged using
4.
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.
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. 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.
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.
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.
2 Texas Red
™
-X Protein Labeling Kit User Guide
Determine the Degree of Labeling (Optional)
1. Measure the absorbance of the conjugate solution at 280 nm
and 595 nm (A
length.
Note: Dilution of the sample may be necessary.
2. Calculate the concentration of protein in the sample:
280
and A
) in a cuvette with a 1 cm path
595
molecules per protein for optimal labeling. For this
reason, we recommend initially performing the reaction
with 0.5 mL of 2 mg/mL protein solution, as described
for IgGs. The labeling conditions can then be optimized
based on the initial results, if desired.
Optimization and troubleshooting
Under-labeling
Where 203,000 is the molar extinction coecient (ε) in
cm-1M-1 of a typical IgG and 0.18 is a correction factor for
the fluorophore's contribution to the absorbance at 280 nm.
3. Calculate the degree of labeling:
Where 80,000 is the molar extinction coecient (ε) in
cm-1M-1 of the Texas Red™-X dye at 595 nm. For IgGs,
optimal labeling is typically achieved with 2-4 moles of Texas
Red™-X dye per mole of antibody
Storing and handling conjugates
Store the labeled protein at 2–8°C, protected from light. If the
final concentration of purified protein conjugate is less than 1
mg/mL, add BSA or other stabilizing protein at 1–10 mg/mL. In
the presence of 2 mM sodium azide, the conjugate should be
stable at 2–8°C for several months. For longer storage, divide
the conjugate into small aliquots and freeze at ≤–20°C. Avoid
repeated freezing and thawing.
It is good practice to centrifuge solutions of conjugates in a
microcentrifuge before use; only the supernatant should then be
used in the experiment. This step will remove any aggregates that
may have formed during storage.
Optimize the kit for use with other proteins
and/or concentrations
• Proteins at less than 2 mg/mL
– Proteins at concentrations less than 2 mg/mL will not
label as eciently. If the protein cannot be concentrated
to ~2 mg/mL, you may wish to use less than 1 mg
protein per reaction to increase the molar ratio of dye
to protein. In addition, using a dilute protein solution,
especially at <1 mg/mL will make it more dicult
to eciently remove the unconjugated dye from the
dye-labeled protein with acceptable yields, since the
provided purification columns are designed to purify
conjugates from a total volume of less than 1 mL. For
reaction volumes greater than 1 mL, you can divide
the solution of the conjugate and apply it to multiple
purification columns or, to avoid further dilution of
the conjugate, you can remove free dye by extensive
dialysis.
• Proteins with molecular weight (MW) other than ~145,000
– Typically, lower MW proteins require fewer dye
molecules and higher MW proteins require more dye
If calculations indicate that the protein is labeled with significantly
less than 2 moles of fluorophore per mole of 145,000–150,000
MW protein, your protein is probably under-labeled. A number of
conditions can cause a protein to label ineciently.
• Trace amounts of primary amine-containing components in
the buer react with the dye and decrease the eciency of
protein labeling. If your protein has been in amine-containing
buers (e.g., Tris or glycine), dialyze extensively versus PBS
before labeling.
• Dilute solutions of protein (≤1 mg/mL) will not label eciently.
• The addition of sodium bicarbonate (step 3) is designed to
raise the pH of the reaction mixture to ~8, because TFP and
succinimidyl esters react most eciently with primary amines
at slightly alkaline pH. If the protein solution is strongly
buered at a lower pH, the addition of bicarbonate will not
raise the pH to the optimal level. Either more bicarbonate can
be added or the buer can be exchanged with PBS, which is
only weakly buered, or with 0.1 M sodium bicarbonate, pH
8.3, by dialysis or other method prior to starting the reaction.
• Because proteins, including dierent antibodies, react with
fluorophores at dierent rates and retain biological activity
at dierent degrees of dye labeling, the standard protocol
may not always result in optimal labeling. To increase the
amount of labeling, you can relabel the same protein sample,
or you can label a new protein sample using less protein or
more reactive dye per reaction. To increase the amount of
dye in the reaction, you can combine the contents of two
vials of reactive dye together. Some researchers obtain better
labeling with overnight incubations at 2–8°C after an initial
incubation of 1 hour at room temperature.
• If the protein did not elute during centrifugation, the
conjugate may be eluted using a high salt concentration
buer (e.g., PBS, 0.3 M NaCl) or 0.2 M, pH 9.4 bicarbonate.
Incubate the column with end-over-end mixing for 1 minute,
then elute conjugate by spinning at 1,000 x g for 2
minutes. We recommend re-purifying the conjugate using
size-exclusion resin, dialysis, or spin filtration.
Texas Red™-X Protein Labeling Kit User Guide 3
Over-labeling
If calculations indicate that the protein conjugate is labeled
with significantly more than 4 moles of fluorophore per mole
of 145,000-150,000 MW protein, your protein is probably overlabeled. Although conjugates with a high number of attached dye
molecules may be acceptable for use, over-labeling can cause
free dye will remain in the conjugate solution after purification.
The presence of free dye, which can be determined by thin layer
chromatography, will result in erroneously high calculated values
for the degree of labeling (“Determine the Degree of Labeling
(Optional)” on page 3). Remaining traces of free dye can be
removed by applying the conjugate to another column or by
extensive dialysis.
aggregation of the protein conjugate and can also reduce the
antibody's specificity for its antigen, both of which can lead to
nonspecific staining. Over-labeling can also cause fluorescence
quenching of the conjugate. To reduce the amount of labeling,
add more protein to your reaction to decrease the molar ratio of
dye to protein or allow the reaction to proceed for a shorter time.
Inecient removal of free dye
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Despite removing most free dye from protein conjugates using
the provided spin columns, it is possible that trace amounts of
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Revision history: Pub. No. MAN0019833
Revision Date Description
A.0 21 January 2021 New manual.
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