R-Phycoerythrin Labeling Kit - NH₂LK23 Product Manual

R-Phycoerythrin Labeling Kit - NH₂ is primarily used for the preparation of R-phycoerythrin-labeled antibody for immunostaining and cellular proteins for tracing. NH₂-Reactive R-Phycoerythrin, a component of this kit, has succinimidyl ester groups, and can easily make a covalent bond with amino groups of IgG or other proteins without any activation process. Filtration Tube included in this kit is used for removing small molecules in the sample protein such as Tris buffer and amine compounds that interfere with the assay or labeling reaction. The maximum excitation and emission wavelengths of the R-phycoerythrin-labeled proteins are 564 nm and 575 nm, respectively. This kit contains all of the necessary reagents for labeling, including the storage buffer for conjugate.

NH2-Reactive R-Phycoerythrin	3 tubes
WS Buffer	4 ml x 1
Reaction Buffer	200 μl x 1
Filtration Tube	3 tubes

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

Caution After a NH2-Reactive R-Phycoerythrin is taken out from the seal bag, keep the unused NH2-Reactive R-Phycoerythrin(s) in the bag, seal tightly and store at -20°C. Store the other components at 0-5°C.

• 10 μl and 200 μl adjustable pipettes
• Incubator (37°C)
• Microcentrifuge
• Microtubes

• 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 R-phycoerythrin labeling, because it might interfere the filtering or 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°C or after returning to room temperature. This phenomenon does not affect the performance.
• This kit includes microtubes containing solutions. Since there is a possibility that the droplets might attach to the inside walls or caps, please shake them down prior to open.

	Maximum excitation wavelength : 564 nm Maximum emission wavelength : 575 nm
Excitation and emission spectra of R-phycoerythrin-labeled protein

Labeling for IgG

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

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

c) NH₂-Reactive R-Phycoerythrin can be hydrolyzed by water. Proceed to Step 6 immediately after the preparation of the NH₂-Reactive R-Phycoerythrin solution.

d) One to two R-phycoerythrin should be introduced into one IgG molecule. Unconjugated R-phycoerythrin remained in the solution might cause background increase with immunoassay. If purification is necessary, purify the conjugate using a gel permeation column or an affinity column for IgG.

e) We recommend using WS Buffer to recover the conjugate. However, you can use an appropriate buffer for the downstream experiments.

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

Yes. However, if antibody solution contains other proteins such as serum albumin or gelatin, labeling reaction might be interfered by that protein.
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 long is the R-phycoerythrin labeled protein stable?

The stability depends on the protein itself. For longer storage, add equal volume of glycerol to the sample solution and store at -20oC.

What is the 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.

Does NH₂-Reactive R-Phycoerythrin form an oligomer during the labeling reaction?

No. Since all amino groups of NH₂-Reactive R-Phycoerythrin are blocked, no oligomerization is occurred.

Can I use the R-Phycoerythrin conjugated protein that is precipitated in storage?

Yes. The precipitated protein should be removed by centrifugation at 10,000 x g for 10 min, and use the supernatant.

Is there any notice for treatment of living cells with the R-Phycoerythrin conjugated protein?

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

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.