CY3 Labeling Kit

The CY3 Labeling Kit from offers all necessary reagents required to perform CY3 labeling of antibodies. It is designed to label antibodies with amidogen (NH2-). The biotin in the kit is sufficient and has been activated for direct use. And the reagents supplied are sufficient to perform 3 labeling reactions each containing 0.1 to 1 mg of antibody or other protein. Each kit includes 6 filtration tubes for desalination of the antibody solution without the need to perform dialysis. The whole procedure is simple and can be completed in 100 min.

Features of the CY3 Labeling Kit

• All-inclusive: The kit provides all the reagents required for CY3 labeling.
• Quick: The whole procedure takes only 100 min.
• Convenient: Desalination can be achieved by filtration, sparing the need for dialysis or gel filtration.
• Flexible: The procedure can be easily adapted to both smaller and larger scales, with 0.1-1 mg of protein labeled each time.
• Perfect results: The kit has been optimized to determine the optimum labeling ratio of CY3 to antibody, lowering the possibility of protein inactivation resulted from excess CY3 labeling.

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Components of the CY3 Labeling Kit

Component	Amount
Reactive CY3	0.15 mg/vial x3
Labeling Buffer	10 mL x1
DMF	500 uL x1
Filtration tube	0.5 mL/tube x6

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Materials required but not included in this kit

• 10uL,50uL,200uL,1000uL adjustable pipettes
• Incubator (37°C)
• Centrifuge (the centrifugal force can be up to 12,000g)

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Storage

The kit is stable for 1 year at 2-8°C before opening.

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Principle of the assay

The CY3 reacts with the primary amine (N-terminal and the side chain of lysine residue) specifically, forming stable amide bond.

 

 

 

 

 

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Calculation on the amount of CY3

The volume of Reactive CY3 used in every reaction depends on the amount and concentration of the protein to be labeled. By optimization, we determine that it’s optimum when the molar ratio of the CY3 to protein is 20:1 in labeling 2 mg/mL of protein sample (IgG, 150 kDa).

• Calculate millimoles of Reactive CY3 to add to the reaction for a 20-fold molar excess:

mL protein*(2 mg protein/ml protein)*(1 mmol IgG/150000 mg IgG)*(20 mmol CY3/mmol protein) = mmol CY4

• Calculate microliters of 10 mM CY3 to add to the reaction:

mmol CY3*(1000000ul/L)*(L/10mmol)=uL CY3

Example: About 13.3uL of 10mM Reactive CY3 solution is to be added for 0.5 mL of 2mg/mL IgG (150,000 MW) solution.

0.5 mL protein*(2mg protein/ml protein)*(1mmol IgG/150000mg IgG)*(20mmol CY3/mmol protein)=0.000133 mmol CY3

0.000133 mmol CY3*(1000000ul/L)*(L/10mmol)=13.3 uL CY3

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Assay protocol

Preparation before experiment:

1. Read the manual carefully.
2. Calculate the volume of Reactive CY3 to be added.
3. Thaw the kit to room temperature 20 min before experiment (Note: put unused Reactive CY3 back to the refrigerator).
4. Dissolve CY3: add 30 uL DMF to the vial of Reactive CY3, let it stand for 10 min until it is dissolved copletely. The concentration of CY3 is now of 10 mM.

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Assay procedure (we label 1mg of protein in this assay)

1. Add 1mg protein sample and corresponded volume of Labeling Buffer to Filtration tube until reaching 0.5 mL. Centrifuge at 12,000 x g for 10min. (Note:A. the maximum volume of Filtration is 0.5mL. B. the protein sample can be under centrifugal ultrafiltration first when at low concentration.)
2. Add 13.3 uL Reactive CY3 and appropriate Labeling Buffer to the Filtration tube, making the final concentration of the protein solution at 2 mg/mL.Pipette to mix it thoroughly and incubate the tube for 60 min at 37°C.
3. Centrifuge at 12,000 x g for 10 min.
4. Add appropriate Labeling Buffer to the Filtration tube until reaching 0.5 mL. Pipette to mix it thoroughly and centrifuge at 12,000 x g for 10 min. Repeat this step once .
5. Add 0. 2 mL Labeling Buffer to the Filtration tube and pipette to mix it thoroughly. Invert the filtration tube and put it into another centrifugal tube. Centrifuge at 6 000g for 10 min.
6. Collect the solution in the centrifugal tube –> this is your CY3-labeled antibody.

 

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Precautions

• This kit can be also used to label antigen,HRP and polypeptides with amidogen (NH2-). The labeling ratio depends on theamount of amidogen.
• DMF should be preserved airtight in a dry place. Seal it with the parafilm immediately after use.
• In the procedure 5 above, Labeling Buffer is used to collect the labeled protein.You can also use other buffer or protective agents as you like.
• The kit is stable for 1 year before opening. Please use it before its expiration date.
• The Filtration tube provided in the kit has a molecular weight cutoff (MWCO) of 10KD. So please be careful of the molecular weight of the antigen or polypeptide to be labeled.
• In the step 2 above, for other quality antibodies, we should control the final concentration of antibody is 2 mg/ml strictly, then calculate the volume of NH2- Reactive CY3 required according to the quantity of the antibodies.
• Ensure no free amino-group being introduced during the coupling process. (Tris, ammonia and sodium azide can react with an activated CY3, thus reducing the conjugation rate of protein-CY3).

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Material and Datasheet