Fluorescein Labeling Kit - NH2 LK01 manual

General Information

Fluorescein Labeling Kit - NH₂ is primarily used for the preparation of fluorescein-labeled antibody for immunostaining and cellular proteins for tracing. NH₂-Reactive Fluorescein, a component of this kit, has a succinimidyl ester group, and can easily make a covalent bond with an amino group of the target protein or other macromolecules without any activation process. Filtration Tube included in this kit is used for sample protein in removing small molecules such as Tris buffer and amine compounds that interfere with the assay or labeling reaction. The labeling process is very simple. Add the NH₂-Reactive Fluorescein to protein solution on a filter membrane, and incubate at 37^oC for 10 min. Excess Fluorescein molecules can be removed by a filtration tube. The excitation and emission wavelengths of the fluorescein-labeled proteins are 500 nm and 525 nm, respectively. This kit contains the necessary reagents for labeling, including the storage buffer for conjugates.

Kit Contents

NH₂-Reactive Fluorescein	3 tubes
WS Buffer	4 ml x 1
Reaction Buffer	500 μl x 1
Filtration Tube	3 tubes

Capacity

Three samples labeling

Sample requirement: Molecular weight > 50,000; amount: 50-200 μg

Storage Condition

Store at 0-5^oC. This kit is stable for 1 year at 0-5^oC before opening.

Caution

After a NH₂-Reactive Fluorescein is taken out from the seal bag, keep the unused NH₂-Reactive Fluorescein(s) in the bag, seal tightly and store at -20^oC.
Store the other components at 0-5^oC.

Required Equipment

10 μl and 200 μl adjustable pipettes
Incubator (37^oC)
Microtubes
Microcentrifuge
DMSO

Precaution

If the target protein solution contains other proteins with molecular weight larger than 10,000 such as serum albumin or gelatin, purify the protein solution, and use the purified target proteins for fluorescein labeling, because it might interfere the labeling reaction.
If the protein solution contains small insoluble materials, centrifuge the solution, and use the supernatant for the labeling.
The droplets which induced from the dry inhibitor of membrane, are occasionally found inside Filtration Tube while storing the kit at 0-5^oC or after returning to room temperature. This phenomenon does not affect the performance.

General Protocol

Step 1.
Add 100 μl WS Buffer and the sample solution containing 50-200 μg protein^a) to a Filtration Tube.

Step 2.
Mix the solution with pipetting several times, and centrifuge at 8,000 x g for 10 min.^b)

Step 3.
Add 10 μl DMSO to NH₂-Reactive Fluorescein, and dissolve with pipetting.^c)

Step 4.
Add 100 μl Reaction Buffer to the Filtration Tube, and then add 8 μl ^d)NH₂-Reactive Fluorescein solution to the Filtration Tube and pipette to mix

Step 5.
Incubate the tube at 37°C for 10 min.

Step 6.
Add 100 μl WS Buffer to the Filtration Tube, and centrifuge at 8,000 x g for 10 min.^b)Discard the filtrate.

Step 7.
Add 200 μl WS Buffer to the Filtration Tube, and centrifuge at 8,000 x g for 10 min.^b) Repeat this step one more time.

Step 8.
Add 200 μl WS Buffer, and pipette about 10 times to recover the conjugate.^e) Transfer the solution to a microtube (not included in this kit), and store at 0-5°C.

a) The volume of protein solution should be less than 100 μl. If the protein concentration is lower than 0.5 mg/ml, repeat Steps 1 and 2 until the total protein accumulation becomes 50 - 200 μg.

b) If the solution still remains on the membrane after the centrifugation, spin for another 5 min.

c) NH₂-Reactive Fluorescein is on the bottom of the tube. Add 10 μl DMSO to the bottom of the tube, and pipette several times to dissolve.
NH₂-Reactive Fluorescein can be hydrolyzed by moisture in DMSO. Proceed to Step 4 immediately after the preparation of the NH₂-Reactive Fluorescein solution.

d) If the amount of protein is 200 μg, add entire NH₂-Reactive Fluorescein solution.

e) We recommend using WS Buffer to recover the conjugate. You can choose any kinds of buffers appropriate for your experiment.

Determination of Fluorescein / protein Ratio

Dilute the fluorescein-labeled protein solution with WS Buffer or other neutral buffer to a proper volume then measure the absorbance of the protein solution at 280 nm and 500 nm, if you need to know the fluorescein/protein ratio. Calculate the ratio using the following equation: When target protein is IgG, use 216,000 as the ε of IgG. Molar absorption coefficient of fluorescein in WS Buffer is 60,000.

Ratio (fluorescein molecules per protein molecule) = A₅₀₀ / 60,000 (A₂₈₀- A₅₀₀ x 0.22) / (ε of protein)

A₅₀₀: absorbance at 500 nm
A₂₈₀: absorbance at 280 nm
ε: molar absorption coefficient of protein at 280 nm

Fluorescein Labeling Reaction

Q & A

Can I use this kit to label antibodies which is commercially available?

Yes. However, if antibody solution contains other proteins such as serum albumin or gelatin, labeling reaction might be interefered by thatprotein. Purification of the antibody solution with affinity chromatography is necessary prior to use this kit.
Contact us for the purification procedure, if you need.

How many fluorescein molecules are introduced to protein?

The number of conjugated fluorescein depends on the protein. In the case of rabbit IgG, 3 to 7 fluorescein molecules conjugate to each protein molecule.

How long is the fluorescein-labeled protein stable?

Stability of conjugate depends on the protein itself. In the case of labeling for rabbit IgG, the labeled IgG is stable at 4^oC for 2 months. For longer storage, add equal volume of glycerol to the sample solution and store at -20^oC.

What is a minimum amount of protein that can be labeled using this kit?

We recommend using 50 μg as a minimum amount. Though 10 μg protein can be labeled using this kit, the background might be increased.

Can I use this kit to label oligonucleotides or oligopeptides?

No. Oligonucleotides and oligopeptides may be too small to retain on the membrane filter of Filtration Tube.

Is there any notice for treatment of living cells with the fluorescein-labeled protein?

We recommend using PBS including 2-10% FBS for preparation of cell suspension to maintain the best cell conditions.

Does recovery buffer (WS Buffer) have harmful effect to living cells?

No. WS Buffer contains stabilizing agent (surfactant) that is controlled of its concentration without cytotoxicity. If you are concerned about the additive in WS Buffer, you can use your own buffer currently used instead of WS Buffer.

Fluorescein Labeling Kit - NH2LK01 Product Manual