Your present position: Home > Resources > Protocols > Labeling

Business hours:

Monday-Friday


After business hours or during company functions when telephones not answered, you may use our Email address or Livechat in the lower right corner to leave a message.


Tel: 1-240-252-7368

      1-240-252-7376

Fax:1-240-252-7376


CY3 Labeling Kit


Author:Elabscience
views:602

Catalog Number: EBC0002  

Introduction    

The CY3 Labeling Kit of Elabscience offering a collection of reagents required for CY3 labeling, is designed to label antibody with amidogen(NH2-). The CY3 in the kit is sufficient and has been activated for direct use. And the reagents are enough for approximately 3 labeling reactions each containing 0.1-1mg of antibody or other protein. Each kit includes 3 Filtration tubes for desaltination of antibody labeling without the need for dialysis. The whole procedure is simple and can be completed in 100min in proficient operation.

Features  

1. All-inclusive: The kit provides all the reagents required for CY3 labeling.

2. Quick:  The whole procedure takes only 100min.

3. Convenient: Desaltination can be achieved by Filtration tube, sparing the need for dialysis or gel filtration.

4. Flexible: The procedure can be easily adapted to both smaller and larger scales, with 0.1-1mg of protein labeled each time.

5. 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.

Components  

 Component

Amount

Reactive CY3

0.15mg/vial×3

Labeling Buffer

10mL×1

DMF

500uL×1

Filtration tube

0.5mL/tube×6

Materials required but not included in this kit  

1. 10μL,50μL,200μL,1000μL adjustable pipettes

2. Incubator(37°C)

3. Centrifuge(the centrifugal force can be up to 12,000×g)

Storage  

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

Principle of the assay  

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

 

Calculation on the amount of Reactive 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 2mg/mL of protein sample(IgG ,150KD).

1.Calculate millimoles of Reactive CY3 to add to the reaction for a 20-fold molarexcess:

mL protein*( 2mg protein/mL protein)*(1mmol IgG/150,000mg IgG)*(20mmol CY3/mmol protein)=  mmol CY3

2.Calculate microliters of 10mM Reactive CY3 to add to the reaction:

mmol CY3*(1,000,000μL/L)*(L/10mmol)=  μL CY3

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

0.5mL IgG*(2mg IgG/1mL IgG)*(1mmol IgG/150,000mg IgG)*(20mmol CY3/1mmol IgG)=0.000133mmol CY3

0.000133mmol CY3*(1,000,000μL/L)*(L/10mmol)=13.3μL CY3 Solution

Assay protocol  

Preparation before experiment:

1.Read the manual carefully.

2.Calculate the volume of Reactive CY3 to be added.

3.Bring the kit to room temperature 20min before experiment (Note: put unused  Reactive CY3 back to the refrigerator).

4.Dissolve  Reactive CY3: add 20μLDMF to the vial of  Reactive CY3, let it stand for 10min until it dissolved fully. The concentration of CY3 is 10mM now.

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 0.5mL. Centrifuge at 12,000 x g for 10min. (Note:①the maximum volume of Filtration is 0.5mL.  ②the protein sample can be under centrifugal ultrafiltration first when at low concentration.)

2.Add 13.3μL  Reactive CY3 and appropriate Labeling Buffer to the Filtration tube, making the final concentration of the protein solution at 2mg/mL.Pipette to mix it thoroughly and incubate the tube for 60min at 37°C.

3.Centrifuge at 12,000 x g for 10min.

4.Add appropriate Labeling Buffer to the Filtration tube until reaching 0.5mL. Pipette to mix it thoroughly and centrifuge at 12,000 x g for 10min. Repeat this step once again.

5.Add 0.2mL 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 6,000× g for 10min.

6.Collect the solution in the centrifugal tube, namely antibody labeled by CY3.

Precautions  

1.This kit can be also used to label antigen, HRP and polypeptides with amidogen(NH2-). The labeling ratio depends on the amount of amidogen.

2.DMF should be preserved airtight in a dry place. Seal it with the parafilm immediately after use.

3.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.

4.The kit is stable for 1 year before opening. Please use it before its expiration date.

5.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.

6.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 Reactive CY3 required according to the quantity of the antibodies.

7.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).