Updated: September 25, 2019

For research use onlyĂ
Table 1. Product information
CAT. No.

Product nameSizè

Storage upon receipt

Stability≤−20°C, keep desiccated1 year (when unopenedand protected from light.

and stored as described.)

1.
SaraFluor 488B (also known as HEtetTFER) is a green fluorescent probe developed exclusively for superresolution imaging. Spontaneous blinking of this fluorophore is suitable for

SMLM (single molecule

localization microscopy) super-resolution imaging. This ability allows to perform the super-resolution imaging
using STORM, PALM and other similar microscopy under physiological solution conditions without addition ofthiols, deoxidizing reagents or exposure to strong laser irradiation to induce blinking as with general fluorescentprobes. Since this product is an NHS compound that forms a covalent bond with amino group only by mixing,
it can be easily used to label proteins and nucleic acids. About 5.5 - 11 nmol of proteins (or about 0.8 - 1.6 mgof IgG antibodies) can be labeled with this product.
Because the blinking frequency of this fluorescent probe is relatively low compared to that of SaraFluor
650B (also known as HMSiR), it is recommended to use higher labeling ratio (3-5) when labeling antibodies.
Table 2. Specifications
λex (nm)*1λem (nm)*2ε (M-1cm-1)*3507

530

80,000*1 The wavelength of maximal excitation*2 The wavelength of maximal emission*3 Molar absorption coefficient at excitation
maximum wavelength■ Storage
This product is shipped as a dried solid in a nitrogengas filled vial. Upon receipt, store at ≤−20°C, keep
desiccated and protected from light. Dissolve theFigure 1. Fluorescence spectra

reagents to DMSO just before the use. We provide
no warranty for the reagents which was stored as asolution.

12.

An example of protein labeling

3. Check the composition of the antibody solution you
wish to label. If the solution contains proteins that are■ Materials required but not provided

not sufficiently purified, BSA stabilizer or other1.

Dimethylsulfoxide, anhydrous (DMSO).

2.

0.1 M Sodium hydrogen carbonate (NaHCO3, pH 8.4).

3.

Phosphate buffered saline (PBS, pH 7.4) or the buffersolution suitable for target proteins.

4.

Acentrifugalultrafiltration

filterforproteinconcentration (eg: Pall Nanosep or Amicon Ultra;
select one with MWCO of 1/3 - 1/6 of molecularweight of target proteins.).
5.

A gel filtration column (eg: GE Healthcare NAP-5,
NAP-10, NAP-25, etc.; size of the column shouldmatch the amount and molecular weight of target
proteins).

6.

A blocking solution (10 mg/mL BSA solution
dissolved in PBS pH 7.4)

G are often used for antibodies. If proteins aredissolved in Tris buffer or other solution containing
primary amines, solution exchange is necessary.
Exchange it with a buffer solution that does not contain
primary amines using a spin column, NAP-5 or othergel filtration column, or dialysis.

Antibody concentration and solution exchange
1. Measure concentration of the purified protein, put theantibody solution into the centrifugal ultrafiltration filter,and concentrate it in the centrifuge to the volume of

2. Concentrate the protein about 10 times in the centrifugal

SaraFluor 488B is yellowish red solid. Before opening
the vial, spin down the solid to the bottom by amicrocentrifuge or by a desktop centrifuge to collectthe solid.

2.

Affinity purification methods using Protein A or Protein

about 1/10.

■ Preparation of reagent
1.

proteins mixed in, the solution requires purification.

Warm the vial to the room temperature and add 11 ̀lof DMSO to one vial to make 10 mM stock solution.

ultrafiltration filter and then add 0.1 M NaHCO3 (pH 8.4)
buffer to restore the original volume. Concentrate thesolution further in the centrifuge until the antibody
concentration becomes 2 mg/mL or more.
The IgG concentration can be easily calculated fromabsorbance at 280 nm (A280) using Eq. 1.

Dissolve the solid completely by careful pipetting.
Solution will be also yellowish red liquid.

3.
̀※ Since some proteins is not stable in the 0.1 M NaHCO3(pH8.4)

buffer,considerappropriate

solutionconditions and act quickly after buffer replacement.

■ IgG antibody labeling example
Labeling preparation
1. Puttheblocking

solution

into

thecentrifugal

ultrafiltration filter and incubate it at the room
temperature for 15 minutes. Remove the blocking
solution and rinse the filter thoroughly with PBS 10̀

times or more. Next, put 200 ̀L of PBS to the filterand centrifuge it to wash the filter.
2. Remove preservation solution from the gel filtration

̀column and equilibrate it thoroughly according to thecolumn manual.
2

#$ % &'&()#+&*

$ %,$ %*-.

/)※■ Fluorescence observation

1. To evaluate the protein concentration, measure theabsorbance at 280 nm (A280) or determine the protein
concentration (Cprotein in mg/mL) by the Bradford
protein assay method.
2.

Table 2. Related Products
CAT. No.

Product name

EAREE Bldg. 5F, Kita 8 Nishi 18-35-100, Chuo-ku, Sapporo 060-0008, JAPANTEL: +81-11-624-5860
https://goryochemical.com/

Description3