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FITC Labeling Kit
Catalog Number: EBF0003
The FITC Labeling Kit of Elabscience offering a collection of reagents required for FITC labeling, is designed to label antibody with amidogen(NH2-). The specially treated FITC in the kit can be used directly. The reagents are enough for approximately 3 labeling reactions each containing 0.1-2mg 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.
1. All-inclusive: The kit provides all the reagents required for FITC 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 0.1-2mg of protein labeled each time.
5. Perfect results: The kit has been optimized to determine the optimum labeling ratio of FITC to antibody, lowering the possibility of protein inactivation resulted from excess FITC labeling.
Materials required but not included in this kit
1. 10μL，50μL，200μL，1000μLadjustable pipettes
3. Centrifuge(the centrifugal force can be up to 12,000×g)
The kit is stable for 1 year at 2-8°C before opening.
Principle of the assay
Protein bonds covalently to FITC by thiourea connection as free amino groups of lysine residues in the protein molecule can react with nucleophilic FITC.
Calculation on the amount of FITC
The volume of FITC used in each reaction depends on the amount and concentration of the protein to be labeled. When labeling 2mg / ml of antibody (IgG, 150KD), taking antibodies and FITC with the mass ratio of 1mg: 150ug can achieve better marking effect.
Preparation before experiment:
1.Read the manual carefully.
2.Calculate the volume of FITC to be added.
3.Bring the kit to room temperature 20min before experiment (Note: put unused FITC back to the refrigerator).
4.Dissolve FITC: add 30μLDMF to the vial of FITC, let it stand for 10min until it dissolved fully. The concentration of FITC is 10mg/ml 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 15μL FITC 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 FITC.
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 FITC 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 FITC, thus reducing the conjugation rate of protein-FITC).