This kit is supplied with FITC that has been pre-activated for direct use, as well as reagents sufficient for approximately 3 labeling reactions of 0.1-2mg each of antibody or other protein. Each kit includes 6 filtration tubes for desalting and thus eliminates the need for dialysis. The whole procedure is simple and can be completed in 100 minutes.
Principle:The isothiocyanate group reacts with primary amines (N-terminal and the side chain of lysine residues) in antibodies and other proteins, forming a stable thiourea bond between the antibody/protein and FITC.
Kit Components:382897A: Activated FITC, 3 x 0.3mg382897B: Labeling Buffer, 1 x 10ml382897C: DMF (Dimethylformamide), 1 x 500ul382897D: Filtration tube (6 tubes of 0.5m capacity per tube)
Materials Required But Not Supplied:Adjustable high-precision transfer pipets (10-1000 uL)Incubator (37°C)Centrifuge (capable of speed up to 12,000 x g).
Storage and Stability:Store kit at 4°C. Unopened kit can be stored at 4°C for one year before opening and should be used before this expiration date. For maximum recovery of product, centrifuge the original vials prior to removing the cap.
Coupling Procedure: The following procedure is based on labeling of 1mg of antibody/protein. Adjust volume of Activated FITC Solution relative to the actual amount of antibody/protein to be labeled.
1. Mix 1mg of protein sample and corresponding volume of labeling buffer to filtration tube to make the total volume equal to 0.5ml. Centrifuge at 12,000 x g for 10 minutes
Note: The maximum volume capacity of the filtration tube is 0.5ml. If the protein concentration in the sample is low, the sample can be concentrated first using centrifugal ultrafiltration (use filters with appropriate molecular weight cut-off).
2. Add 15ul of Activated FITC and appropriate volume of Labeling Buffer to the filtration tube to make the final concentration of the protein solution equal to 2mg/ml. Mix thoroughly using a pipet and incubate the tube for 30 minutes at 37°C.
3. Centrifuge at 12,000 x g for 10 minutes.
4. Add appropriate volume of Labeling Buffer to the filtration tube to make the total volume equal to 0.5ml. Mix thoroughly using a pipet and centrifuge at 12,000 x g for 10 minutes.
5. Repeat step 4.
6. Add 0.2ml of Labeling Buffer to the filtration tube and mix thoroughly using a pipet. Invert the filtration tube into another centrifugal tube. Centrifuge at 6,000 x g for 10 minutes.
7. Collect the solution in the centrifugal tube; this liquid will contain the FITC-conjugated antibody.
