For research use only

Updated: September 25, 2019Ă
1. About SaraFluor-NHS

Table 1. Product information

SaraFluor series is a product line of bright fluorophores,

Product namewhich are suitable for labeling proteins (e.g., antibodies)and other macromolecules. These products are also used
for labeling or marking to evaluate physical properties.

SaraFluor
488-NHS

Among them, SaraFluor 650, 700, 720, which areAmount

Catalog no.

5 nmol × 5

ST1003-10

1 mg

ST1003-11

1 mg × 5

ST1003-15

5 nmol × 5

ST1006-10

1 mg

ST1006-11

1 mg × 5

ST1006-15

5 nmol × 5ST1008-10

1 mg

ST1008-11

1 mg × 5

ST1008-15

5 nmol × 5

ST1010-10

1 mg

ST1010-11

1 mg × 5

ST1010-15

5 nmol × 5

ST1011-10

1 mg

ST1011-11

1 mg × 5

ST1011-15

derivatives of silicone rhodamine, are uniquely bright andphotostable fluorophores in far-red to near-infrared
wavelength range. Thus, they possess outstanding

SaraFluor

brightness and photostability.

600-NHSIn

mildlyalkaline

solutions(pH8.3-8.5),

N-hydroxysuccinimide (NHS) esters quickly form covalentbonds with primary amines (e.g., in side chains of lysineresidues or N-terminal amino groups of peptides).
Therefore, SaraFluor-NHS provides simple labeling ofSaraFluor
650-NHS

proteins and macromolecules just by mixing.

SaraFluor■ Storage

700-NHSThis product is shipped as a solid in a nitrogen gas-filledvial. Upon receipt, store the product desiccated andprotected from light at ≤ −20°C. A shelf-life is 1 year when
it is unopened and stored as previously described.
Dissolve the reagents to DMSO just before the use. We
provide no warranty for the reagents which was stored as

SaraFluor
720-NHSa solution.

※5 mg packages were previously shipped as a single 5mg vial. New lots are shipped as 1 mg × 5 vials. For
the time being, both packagings might be mixed. We
appreciate your understanding.

12.

Spectra and properties of SaraFluor series

Table 2. Properties of SaraFluor-NHS
Fluorophore

SaraFluor 488
SaraFluor 600
SaraFluor 650
SaraFluor 700
SaraFluor 720

Solid color/DMSO

Solution volume per

λex

λem

ε (M-1cm-1)

CF280

solution colordye 1 mg (10 mM)(nm) *1(nm) *2*3*4

Orange/Yellow

209 µL494514

0.82 x 105

0.25

607

1.3 x 1050.18660

1.1 x 105

0.09712

1.0 x 105

0.07740

1.6 x 105

0.14Brown/Purple
Blue/BlueDark blue/Light blue
Green/Light blue-green

180 µL153 µL148 µL127 µL

597
646
691
721

*1 Maximum excitation wavelength*2 Maximum emission wavelength*3 Molar extinction coefficient*4 Correction factor for calculating labeling ratio

3.

An example protocol of protein labeling■ Materials required but not provided
・Anhydrous dimethyl sulfoxide (DMSO)

・

0.1 M sodium bicarbonate buffer (NaHCO3, pH 8.4)

・An appropriate elution buffer (e.g., PBS, pH 7.4, orother buffer solutions suitable for the target protein)・■ Preparation of the reagent
1.

before opening the cap. The compounds might
adhere to the cap or the wall.
2.

Reaction with water could degrade the product anddesalting (e.g., Pall Nanosep or Amicon Ultra; Use thedecrease its reactivity. Dissolve the powder justunit which has an MWCO by 1/6 to 1/3 of molecular・A gel filtration column (e.g., NAP-5, NAP-10, NAP-25(GE Healthcare); Please choose the column byreferring to the column capacity, the amount ofproteins, etc.)

・Blocking solution (e.g. 10 mg/mL BSA solution in PBS,pH 7.4)

Warm the vial to the room temperature before
opening the cap, to avoid moisture adsorption.

A centrifugal filter unit for concentrating proteins orweight.)

Spin-down the compound by using a microcentrifuge

before use.
3.

Dissolve the compound with DMSO at the volume
shown in “Solution volume per dye 1 mg (10 mM)”
from Table 2. Mix the solution thoroughly by pipetting.
The final concentration is 10 mM. For 5 nmol vial,dissolve the solid with 5 µL of DMSO to make 1 mMstock solution.
2

※ Dissolve the dye with DMSO at 10-fold volume to makea centrifugal filter unit. Next, recover the volume by

1 mM stock solution if the dye dissolves poorly.

adding 0.1 M NaHCO3 buffer. Quickly label theprotein after the buffer exchange because some

■ Protein labelingproteins are not stable in the pH 8.4 buffer.

Preparation before labeling
1. Add blocking solution to the centrifugal filter unit andProtein labelingleave for 10-30 min at room temperature. Then,

1. Add the stock solution of SaraFluor-NHS to the protein

remove the blocking solution and rinse the filter unit

solution and mix well immediately by gentle pipetting.

with elution buffer more than 5 times. After that, add

The molar ratio of the fluorophore to the protein

elution buffer to the filter unit, centrifuge the filter unit

should be adjusted to 2–4. Typically, 2.5-fold of

to let the buffer flow through the filter unit.

fluorophore gives appropriate labeling ratio. However,

2. Discard the preserving solution from the gel filtration
column and equilibrate the column with the elution
buffer according to the manufacturer's protocol.

the molar ratio should be adjusted depending on thetarget protein.
2. Incubate the reaction mixture at 37°C for 0.5–1 hourin the dark. Mix the tube by tapping every 10–15 min.

Protein purification, concentration and buffer exchange

3. Add the mixture to the equilibrated gel filtration column.

1. Protein purification before the labeling is required if

Elute with the elution buffer to separate the labeled

the target protein has not been purified enough, or ifprotein with the unconjugated free fluorophores.

amine-containing

protein

Measure the A280 of each fraction to calculate thestabilizers (e.g., BSA) has been added. Use anprotein concentration. Collect fractions which containappropriate protein purification method. For example,

thean affinity purification method utilizing either protein

unconjugated fluorophores.

additives

includingprotein.

Discardfractionscontaining

A or protein G is commonly used for IgG purification.

4. Concentrate on the labeled protein if necessary.

Additionally, buffer exchange is necessary when theMeasure the absorbance and calculate a labelingsolution contains primary amines (e.g., Tris buffer).

ratio (r) byUse either spin columns, gel filtration columns, or̀$dialysis to exchange the amine-containing buffer toan appropriate buffer such as PBS.

A280, Aλex:

2. Concentrate the solution by a centrifugal filter toobtain a protein of 1–3 mg/mL. Measure theCF280:
ɛST:

absorbance at 280 nm (A280) and calculate theconcentration(Cprotein)

byusing

thefollowingequation,(̀)*+absorbance at 280 nm and that at λexcorrection factor at 280 nm (see Table 2.)
molar extinction coefficient of the fluorophore(see Table 2.)

ɛprotein:

molar extinction coefficient of the protein at
280 nm■ Fluorescence observation
MWprotein: molecular weight of protein;

Observe the fluorophore using an appropriate fluorescentimager. For fluorescence microscopy of SaraFluor 488,

ɛprotein:

molar extinction coefficient of protein at

use blue-excitation filter sets for FITC or GFP can be used.

280 nm; 210,000 M-1cm-1 for IgG; 43,800

For SaraFluor 600, yellow-excitation filter sets for Texas

M-1cm-1 for BSA.

Red, etc. can be used. Filter sets for Cy5, Cy5,5 and Cy7
can be used for SaraFluor 650, 700 and 720, respectively.

3. Exchange the buffer to 0.1 M NaHCO3 buffer (pH 8.4)
as follows. Concentrate the protein by 10-fold using

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