Long-arm Biotin Labeling Kit – 3 rx
Sufficient to perform 3 labeling reactions each containing 0.1 to 2 mg of antibody or other protein.
0 in stock (expect 7-14 days for delivery)
0 in stock (expect 7-14 days for delivery)
Long-arm Biotin Labeling Kit
The Long-arm Biotin Labeling Kit is designed to label antibody with amidogen (NH2-). Long-arm reduces the steric hindrance when several biotinylated molecules combine with the same avidin molecular compound, effectively optimizes the labeling and detection results. The biotin in the kit is sufficient and has been activated for direct use. The reagents are sufficient for approximately 3 labeling reactions each containing 0.1 to 2 mg of antibody or other protein. Each kit includes 6 Filtration tubes for desalination of antibody labeling without the need for dialysis. The whole procedure is simple and can be completed in 90 min in proficient operation.
Features of the Long-arm Biotin Labeling Kit
- All-inclusive: The kit provides all the reagents required for biotin labeling.
- Quick: The whole procedure takes only 90 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 0.2-2 mg of protein labeled each time.
- Perfect results: The kit has been optimized to determine the optimum labeling ratio of biotin to antibody, lowering the possibility of protein inactivation resulted from excess biotin labeling.
Components of the Long-arm Biotin Labeling Kit
|NH2-Reactive Biotin||0.17 mg/vial x3|
|Labeling Buffer||10 mL x1|
|DMF||500 uL x1|
|Filtration tube||0.5 mL/tube x6|
Chemical Structure of Long-arm Biotin
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)
The kit is stable for 1 year at 2-8°C before opening.
Principle of the assay
The NH2-Reactive Long-arm 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, 150 kDa).
- 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
- Calculate microliters of 10 mM 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
Preparation before experiment:
- Read the manual carefully.
- Calculate the volume of NH2-Reactive Biotin to be added.
- Thaw the kit to room temperature 20 min before experiment (Note: put unused NH2- Reactive Biotin back to the refrigerator).
- 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 now of 1 0mM.
Assay procedure (we label 1mg of protein in this assay)
- 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.)
- Add 13.3 uL NH2-Reactive Biotin 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 30 min at 37°C.
- Centrifuge at 12,000 x g for 10min.
- 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 .
- 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 6,000g for 10 min.
- Collect the solution in the centrifugal tube –> this is your biotin-labeled antibody.
- 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 Biotin required according to the quantity of the antibodies.
3 rx x 0.1-2 mg antibody labeling