FITC

FITC (Fluorescein Isothiocyanate), is one of the green fluorescein derivatives widely used in biology. FITC has the characteristics of high absorptivity and excellent fluorescence quantum yield. The isothiocyanate group of FITC can be combined with amino, sulfhydryl, imidazole, tyrosyl, carbonyl and other groups on the protein, so as to achieve protein labeling including antibodies and lectins. In addition to its use as a protein marker, FITC can also be used as a fluorescent protein tracer to rapidly identify pathogens by labeling antibodies, or for microsequencing of proteins and peptides (HPLC). The maximum excitation wavelength of FITC is 494 nm. Once excited, it fluoresces yellow-green at a maximum emission wavelength of 520 nm. In addition, FITC is also a hapten that can induce contact hypersensitivity (CHS) and induce an atopic dermatitis model^[1][2][3][4]. In Vitro:Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Protein Preparation
To obtain the best labeling effect, please prepare the protein (antibody) concentration to 1 mg/mL.
1.1 The pH value of the protein solution should be 8.5 ± 0.5. If the pH is lower than 8.0, adjust it with 1 M sodium bicarbonate.
1.2 If the protein concentration is lower than 1 mg/mL, the labeling efficiency will be greatly reduced. To obtain the best labeling efficiency, it is recommended that the final protein concentration range be 1-10 mg/mL.
1.3 The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, otherwise the labeling efficiency will be affected.
2.Dye Preparation
Add anhydrous DMSO into the vial of FITC to make a 1 mg/mL stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of FITC required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of FITC to protein is about 10.
Example: assuming the required marker protein is 1 mL 2 mg/mL IgG (MW=150,000), use 1 mL DMSO dissolve 1 mg FITC, the required FITC volume is 40 μL.
4. Running the Coupling Reaction
1) Slowly add the calculated volume of freshly prepared 10 mg/mL dye to 0.5 mL of protein sample solution, gently shake to mix, and then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent denaturation and inactivation of the protein sample.
2) Place the reaction tube in a dark place and incubate gently with shaking for 60 minutes at room temperature. Every 10-15 minutes, gently invert the reaction tube several times to thoroughly mix the reactants and improve labeling efficiency.
5. Purification of Conjugates
The following protocol is an example of purifying dye-protein conjugates using a Labelling Kits Centrifugation-Based Rapid Desalting Column (5KD) (HY-D3014).
5.1 Prepare the desalting column according to the instructions.
5.2 Load the reaction mixture onto the top of the desalting column.
5.3 Once the sample has run below the resin surface, immediately add PBS (pH 7.2-7.4).
5.4 Add more PBS (pH 7.2-7.4) to the target sample to complete column purification. Collect the fraction containing the desired dye-protein conjugate.