Thermo Fisher Scientific SAIVI User Manual

USER GUIDE

SAIVI™ Alexa Fluor™ 647 Antibody/Protein Labeling Kit

Catalog Number S30044

Pub. No. MAN0019834 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 SAIVI™ Alexa Fluor™ 647 Antibody/Protein 1 mg-Labeling Kit provides a convenient means to label proteins with Alexa Fluor™ 647
near-IR emitting dye (Figure 1). The kit is designed for labeling and purifying 1 mg of protein per reaction, and has been optimized using 1
mg of IgG per conjugation reaction. Comparable amounts of other proteins (>20 kDa) can also be labeled. For labeling smaller amounts of
proteins (20–100 μg), we recommend the Alexa Fluor™ 647 Microscale Protein Labeling Kit (Cat. No. A30009).

To conveniently control the average number of fluorescent dye molecules that become covalently attached to each protein molecule
(the degree of labeling, or DOL), this kit includes a DOL modulating reagent and instructions for decreasing the DOL from its intrinsic
highest value by adding specific amounts of DOL modulating reagent to the labeling reaction. Using this method, you can quickly and
reproducibly obtain protein preparations with varying ratios of dye to protein without significantly altering the labeling conditions or the
purification procedure (Figure 1), allowing more ecient optimization in applications such as in vivo imaging, where the DOL of a protein
can have significant eects on factors such as signal-to-background, biodistribution, and blood clearance.

Contents and storage

Material Amount Storage

Alexa Fluor™ 647 Reactive Dye (Component A) 3 vials (each containing a magnetic

Sodium bicarbonate (MW=84) (Component B) 84 mg

Purification columns (Component C)

DOL modulating reagent, lyophilized solid
(Component D)

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.

• The presence of significant concentrations of proteins other
than the intended labeling target in the solution (e.g., BSA
or gelatin carrier in antibody preparations) will likely result in
poor labeling, due to competition eects.

stir bar)

3 each

1 vial

• Store at 2–6°C
protected from light.

• Do not freeze.

• If the protein is in or has been lyophilized from 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.

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

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. Prepare a solution of DOL modulating reagent. Briefly
centrifuge the DOL modulating reagent (Component D) to
collect any solid at the bottom of the vial. Add 330 µL of
sterile deionized water, cap the tube tightly, and vortex.

3. Prepare 0.5 mL of a ~2 mg/mL protein solution by diluting
it with an appropriate buer and add sodium bicarbonates
as directed here, depending on the nature of the starting
protein sample.

• For protein samples already in a volume of appropriate
buer, add 1/10 volume of 1 M sodium bicarbonate to
the ~500 µL of protein sample (at 2 mg/mL).

• For protein samples lyophilized from an appropriate
buer, prepare a 2 mg/mL solution of the protein by
adding a sucient volume of 0.1 M sodium bicarbonate
buer. (Prepare 0.1 M sodium bicarbonate buer by
diluting the 1 M solution from step 1 10-fold with
deionized water.)

Purify the labeled proteins

Thermo Scientific™ Zeba™ Dye and Biotin Removal Spin Columns
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 buer conditions are used.

IMPORTANT! Protein conjugates that are between 20–50 kDa

require a more alkaline buer system to elute and will retain on the
column if the buer 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.

4. If desired, add an appropriate volume of DOL modulating
reagent solution to the protein vial and mix gently but
thoroughly. To decrease the DOL by ~40%, add 15 µL of
the DOL modulating reagent solution prepared in step 2.
To decrease the DOL by ~70%, add 50 µL of the DOL
modulating reagent solution.

5. Transfer the protein solution to a vial of Alexa Fluor™ 647
reactive dye. This vial contains a magnetic stir bar. Gently
pipette the solution up and down to fully dissolve the
dye (excess agitation of the solution can result in protein
denaturation.

6. Stir the solution for ~1 hour at room temperature, protected
from light. You can perform labeling at lower temperatures,
if necessary; labeling overnight on ice usually yields results
similar to those achieved by labeling for 1 hour at room
temperature. DOL control using the DOL modulating reagent
works similarly at either temperature.

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 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!

centrifugation can result in reduced small molecule removal.

4. If desired, the resin storage buer can be exchanged using
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.

Improper orientation of the column during

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).

Figure 1 Modulation of the degree of labeling achieved
using the DOL modulating reagent. Observed labeling is
60% and 30% of the unmodulated value using 15 μL or 50
μL DOL modulating reagent, respectively. Labeling results
obtained at room temperature for 60 minutes or on ice for
20 hours are essentially identical.

2 SAIVI

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.

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Alexa Fluor™ 647 Antibody/Protein Labeling Kit User Guide