Allophycocyanin Labeling Kit - SHLK24 Product Manual

Allophycocyanin Labeling Kit - SH is primarily used for the preparation of allophycocyanin-labeled antibody for immunostaining and cellular proteins for tracing. SH-Reactive Allophycocyanin, a component of this kit, has maleimide group that reacts with sulfhydryl groups (SH) of reduced IgG or other proteins. This kit contains all of the necessary reagents for the labeling. Reducing Agent in this kit produces free sulfhydryl groups in the IgG molecule without loss of antibody affinity. The labeling process is simple. After the reducing reaction, add SH-Reactive Allophycocyanin to IgG solution on the membrane of Filtration Tube, and incubate at 37oC for 1 hour. The maximum excitation and emission wavelengths of the allophycocyanin-labeled proteins are 650 nm and 660 nm, respectively. This kit contains all of the necessary reagents for labeling, including the storage buffer for conjugates.

SH-Reactive Allophycocyanin	3 tubes
RA Solution	1 ml x 1
WS Buffer	4 ml x 1
Reducing Agent	3 tubes
Reaction Buffer	200 μl x 1
Filtration Tube	3 tubes

Three samples labeling

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

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

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

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

• If the target protein has sulfhydryl groups, skip the reducing procedure(Step 3-6). 
• 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 allophycocyanin 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 amount of Reducing Agent is optimized for the preparation of the reduced IgG. Please examine the necessary amount of Reducing Agent for the reduction of other proteins.
• The droplets which induced from the dry inhibitor of membrane, are occasionally found inside Filtration Tube while storing the kit at 0-5oC 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: 650 nm Maximum emission wavelength : 660 nm
Excitation and emission spectra of Allophycocyanin-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) SH-Reactive Allophycocyanin is unstable in Reaction Buffer. Proceed to Step 8 immediately after the preparation of the SH-Reactive Allophycocyanin solution.

d) One to two allophycocyanin should be introduced into one reduced IgG molecule. Unconjugated allophycocyanin 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 storage the conjugate. You can choose any kinds of buffers appropriate for your experiment.

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.

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.

Can I use the Allophycocyanin 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 Allophycocyanin 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.