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


Biotin Labeling


Author:Elabscience
views:674

Catalog Number: EBLK0002  

Introduction    

The Biotin Labeling Kit of Elabscience offering a collection of reagents required for biotin labeling, is designed to label antibody with amidogen(NH2-). The biotin in the kit is sufficient and has been activated for direct use. And the reagents are enough for approximately 3 labeling reactions each containing 0.2-2mg of antibody or other protein. Each kit includes 6 Filtration tubes for desaltination of antibody labeling without the need for dialysis . The whole procedure is simple and can be completed in 90min in proficient operation.

Features  

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

2. Quick:  The whole procedure takes only 90min.

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 0.2-2mg of protein labeled each time.

5. Perfect results: The kit has been optimized to determine the optimum labeling ratio of biotin toantibody, lowering the possibility of protein inactivation resulted from excess biotin labeling.

Components  

 Component

Amount

NH2-Reactive Biotin

0.1mg/vial×3

Labeling Buffer

10mL×1

DMF

500uL×1

Filtration tube

0.5mL/tube×6

Materials required but not included in this kit  

1. 10uL,50uL,200uL,1000uL 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 NH2-Reactive Biotin reacts with the primary amine (N-terminal and the side chain of lysine residue) specifically, forming stable amide bond.

Calculation on the amount of NH2-Reactive Biotin

The volume of NH-Reactive Biotin 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 Biotin to protein is 20:1 in labeling 2mg/mL of protein sample(IgG,150KD).

1.Calculate millimoles of NH2-Reactive Biotin to add to the reaction for a 20-fold molar excess:

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

2.Calculate microliters of 10mM NH2-Reactive Biotin to add to the reaction:

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

Example: About 13.3uL of 10mM NH2-Reactive Biotin 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 Biotin/mmol protein)=0.000133 mmol Biotin

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

Assay protocol  

Preparation before experiment:

1.Read the manual carefully.

2.Calculate the volume of NH2-Reactive Biotin to be added.

3.Bring the kit to room temperature 20min before experiment (Note: put unused NH2- Reactive Biotin back to the refrigerator).

4.Dissolve NH2- Reactive Biotin: add 30uL DMF to the vial of NH2- Reactive Biotin, let it stand for 10min until it dissolved fully. The concentration of biotin 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: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 13.3uL NH2-Reactive Biotin 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 30min 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 onceagain.

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 Biotin.

Precautions  

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

2.DMFshould 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 NH2- Reactive Biotin required according to the quantity of the antibodies.