Question

5.How to prepare a protein-fluorescein conjugate from FITC?

Answer 1

Fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is the most widely used fluorescent probe for the preparation of conjugates with biological molecules.

FITC is one of the most popular fluorescent probes ever created. An isothiocyanate derivative of fluorescein is synthesized by modification of its lower ring at the 5- or 6-carbon positions. The two resulting isomers are nearly identical in their reactivity and spectral properties, including excitation and emission wavelengths and intensities. Their chemical differences, however, may affect the separation of modified proteins from excess reagent or the analysis of tagged molecules by electrophoresis. For this reason, most manufacturers purify the carbon-5 derivative as the FITC reagent of choice.

Isothiocyanates react with nucleophiles such as amines, sulfhydryls, and the phenolate ion of tyrosine side chains. The only stable product, however, is with primary amine groups, and so FITC is almost entirely selective for modifying ε- and N-terminal amines in proteins . The reaction involves attack of the nucleophile on the central, electrophilic carbon of the isothiocyanate group. The resulting electron shift creates a thiourea linkage between FITC and the protein with no leaving group.

The level of fluorescein modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic excitation maximum (~498 nm). The number of fluorochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is important in predicting the behavior of antibodies labeled with FITC . Using the known extinction coefficient of FITC in solution at pH 13 (ε498 nm=8.1–8.5×104) a determination of derivatization level can be made after excess FITC is removed. At pH 7.8, the absorbance of protein-coupled FITC decreases by 8%.

PROTOCOL :-

1.

Prepare a protein solution in 0.1-M sodium carbonate, pH 9.0, at a concentration of at least 2 mg/ml.

2.

In a darkened lab, dissolve FITC (Thermo Fisher) in dry DMSO at a concentration of 1 mg/ml.

3.

In a darkened lab, slowly add 50 to 100 μl of FITC solution to each milliliter of protein solution (at 2-mg/ml concentration). Gently mix the protein solution as the FITC is added.

4.

React for at least 8 h at 4°C in the dark.

5.

The reaction may be quenched by the addition of ammonium chloride to a final concentration of 50-mM.

6.

Purify the derivative by gel filtration using a PBS buffer or another suitable buffer for the particular protein being modified. The use of a desalting resin with low exclusion limits works well. To obtain complete separation, the column size should be 15 to 20 times the size of the applied sample.