Updated: September 25, 2019

For research use onlyĂ
Table 1. Product information
Catalog no.

Product nameAmount

A208-01

SaraFluor
650B-NHS

100 μgStorage upon receipt≤−20°C,

keep

desiccatedand protected from light.

Stability
1 year (when unopenedand stored as described.)

1.
SaraFluor 650B-NHS is a fluorescent probe for super-resolution microscopy. Since this probe blinks
spontaneously under physiological conditions, it can be used for single molecule localization microscopy using
microscopes for PALM and STORM. For the observation, neither activation laser irradiation, nor additions ofthiols/reducing reagents are required.
SaraFluor 650B-NHS forms a covalent bond with primary amines in antibodies, other proteins, or othermolecules. Condensing reagents are not necessary for the reaction. This manual describes a protocol for
conjugating SaraFluor 650B-NHS to about 5 mg of IgG antibody.
Table 2. Properties of SaraFluor 650B determined
in 0.1M citrate buffer (pH 3.5).
λex (nm)

λem (nm)ε (M-1cm-1)

654

669

1.2 × 105

■ Storage
This product is shipped as a solid in a nitrogen gas-filledvial. Upon receipt, store the product desiccated andprotected from light at ≤ −20°C. Dissolve the reagents toDMSO just before the use. We provide no warranty forFig. 1. Excitation (red) and emission (magenta) ofSaraFluor 650B-NHS conjugated with a protein. The
spectrum was measured in 0.1 M citrate buffer (pH3.5).

the reagents which was stored as a solution.

1

2.

Protocol for IgG labeling

NHS (30 nmol) to 500 μL of 3 mg/mL IgG (MW~150kDa, 10 nmol; 3:1 in molar ratio). Mix gently with■ Materials required but not provided

pipetting (no vortexing) and incubate for 30 minutes

・Anhydrous dimethyl sulfoxide (DMSO, anhydrous )・Labeling buffer (0.1 M

at 37oC in the dark.

5. Load the dye-protein mixture to the gel filtration

・

column and elute with elution buffer, according to
the instruction of the column. SaraFluor 650B-

・

labeled protein appears first, followed by unreacteddyes. Separate the protein from the unreacted

・Gel filtration column (e. g. NAP-5, GE Healthcare)
・Blocking solution (10 g/mL BSA in PBS)

SaraFluor 650B-NHS by fractionation.

6. Typically, the labeling ratio of the protein is about
0.9—1.8 in this condition. Add an equal volume of■ Preparation of the reagent

glycerol (final, 50%) and keep the solution at -20oC

1.

SaraFluor 650B-NHS is pale blue solid, but it could

until use. Freezing the solution below －25 oC is not

be invisible because of its small amount. Warm therecommended.

vial to the room temperature before opening the cap

2.

to prevent moisture absorption. Centrifuge before■ Calculation of the labeling ratio

opening the cap to prevent the solid adhering to theIn a neutral aqueous solution, SaraFluor 650B molecule iscap. Add 18 μL of anhydrous DMSO to one vial toin an equilibrium between closed form (non-fluorescent)prepare 10 mM stock solution. Dissolve the solidand open form (fluorescent). For accurate measurement ofcompletely through careful pipetting.

the SaraFluor 650B concentration, absorbance should be

Use the solution immediately. Reactivity of NHS tomeasured in pH 3.5 citrate buffer (in which most ofamines is reduced by moisture absorption.

SaraFluor 650B is in the open form) as the following
procedure.

■ Procedure for labeling reaction
1.

First, measure the molar concentration of the protein(Cprot) by BCA method or by Lowry method. Next, dilute theSaraFluor 650B-labeled protein to 0.1M citrate buffer (pH

2. Remove the reservation buffer from the gel filtration

3.5) in a volume ratio of >30 (=d) and measure thecolumn, add blocking solution, close the outlet of theabsorbance at 654 nm (A654). Calculate the labeling ratio

column, and incubate for 15 min at room

using the following equation and the value shown in Table

temperature (RT). Then open the outlet and elute2.

with > ×10 volumes of the column to remove BSA.
This blocking may increase the final protein yield.

3. Condense the target IgG to about ×10 concentrationof the original solution using a centrifugal filter byfollowing the instructions of the filter. Add thelabeling buffer to the original volume and mix well.
Centrifuge the solution again to obtain >1.5 mg/mL
protein solution. Note: The target protein to belabeled should not be in amine-containing buffers
such as Tris.

4. Add SaraFluor 650B to the protein which molar ratio
should be 3:1—5:1 (SaraFluor 650B-NHS: protein).
For example, add 3 μL of 10 mM SaraFluor 650B-■ Microscopy
It is convenient to use microscopes for PALM or STORM
for the observation of SaraFluor 650B. Excitation laser
power of about 30% that is usually used for Alexa Fluor
647 observation is enough (100 W/cm2). Optical filter set
which is used for Alexa Fluor

647 can be used (e. g.

692/40 nm bandpass emission filter, Semrock). Turn off
405 laser which is necessary for observation of Alexa
Fluor 647. SaraFluor 650B can be observed in PBS orother physiological buffer conditions. Addition of antifade
reagents is recommended to reduce photobleaching.

2Following the instructions of the microscope, capture
hundreds to 10-thousands of images and process theseimages to obtain a superresolution image.

Table 3. Related Products
Catalog no.

Product name

Major applications
For superresolution imaging of-labeled proteins.

Superresolution microscopy by immunocytochemistry.

Superresolution microscopy by immunocytochemistry.

Superresolution microscopy by immunocytochemistry.
Superresolution imaging probes which can label thiols. In proteins, it can label
cysteine residues.
For STED imaging of-labeled proteins.

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