Thermo Fisher Scientific Texas Red User Manual

USER GUIDE

Texas Red™-X Protein Labeling Kit

Catalog Number T10244

Pub. No. MAN0019833 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 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 sucient 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 eciently 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 eciency, the purified protein must be in
a buer free of ammonium ions or primary amines.

• If the protein is in an unsuitable buer (e.g., Tris or glycine),
the buer should be replaced with phosphate-buered 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 buer
(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 eciently 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 eciency 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 buer. 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 buer can be exchanged using

4.

a buer of choice. To exchange, add 2 mL of equilibration
buer 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 buer exchange is required to
ensure conjugate recovery.

1. Following storage buer removal, apply 500 µL of 0.2 M, pH

9.4 bicarbonate buer 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 buer
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 buer (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