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Fluorescein Isothiocyanate

Catalog Number: 100276
Fluorescein Isothiocyanate


Structure:



Molecular Formula: C21H11NO5S

Molecular Weight: 389.4

CAS #: 3326-32-7

Form: Isomer I

Synonyms: Fluorescein-5-Isothiocyanate; FITC

Fluorescence: Conjugated FITC has an excitation maximum of 492 nm. It fluoresces with an apple-green light with maximum emission at 515 nm. Extinction Coefficient: 75,000.

Solubility: Soluble in DMF, DMSO (5 mg/ml), acetone (1 mg/ml - clear, yellow solution), ethanol (20 mg/ml) and in aqueous solution pH > 6. Do not store FITC in aqueous media because it is unstable in water. Keeps solutions aliquoted and stored at -20°C and protected from light. Dilute in aqueous buffers immediately before use.

Description: A amine-reactive fluorescein derivative suitable for protein labeling. Attaches to the protein via an amine group.

Adsorbing FITC onto celite has been reported to increase the efficiency of dispersing FITC in a protein solution.11 FITC on celite reportedly reacts very quickly with proteins,11 so much faster that antibody titer may be lost by overacylation of the free amino groups. The FITC on celite does permit weighing manageable quantities when working with small amounts of protein and avoids the use of organic solvents. FITC on celite has been used for the labeling of fibrinogen.14
Note: FITC contributes partial negative charges at normal pH. Antibodies and fragments over-conjugated with FITC can have net negative charges which result in high background fluorescence because of nonspecific bindings to positive surfaces.

Typical Procedure for Labelling Protein with FITC5,6

1. Prepare a solution of at least 2 mg/ml of protein in 0.1 M sodium carbonate buffer, pH 9. Do not store sodium bicarbonate buffer more than 1 week at 2-8°C. The pH of the buffer may change upon storage. It is advised that fresh buffer be made just before use. The protein to be conjugated should be free of contaminating proteins, and protein solutions should not be prepared in buffers containing sodium azide or amines such as Tris or glycine since they inhibit the labeling reaction. If the buffer contains amines or sodium azide, dialyze protein solution against PBS, pH 7.4, overnight at 2-8°C. Avoid dialysis at high pH values (> 8.0) as this may be harmful to some proteins.

2. Dissolve the FITC in anhydrous DMSO at 1 mg/ml. For optimal results, this should be prepared fresh for each labeling reaction.

3. For each 1 ml of protein solution, add 50 ul of FITC solution, very slowly in 5 ul aliquots while gently and continuously stirring the protein solution.

4. After all of the required amount of FITC solution has been added, incubate the reaction in the dark for 8 hours at 2-8°C.

5. Add NH4Cl to a final concentration of 50 mM and incubate for 2 hours at 2-8°C.

6. Add xylene cyanol to 0.1% concentration and glycerol to 5% concentration.

7. Separate the unbound FITC from the conjugate by gel filtration using a fine-sized gel matrix with an exclusion limit of 20,000 to 50,000 (for globular proteins such as antibodies). With the column flow stopped, carefully layer the reaction mixture onto the top of the column. Then open the column, allowing the reaction mixture to flow into the column. Just as it all enters the column bed, carefully add PBS to the top of the column and connect to a buffer supply.


8. Store the conjugate at 2-8°C in the column buffer in a light-proof container. Sodium azide may be added as a preservative (final concentration 0.05%). If the protein concentration is loq (< 1 mg/ml), bovine serum albumin may be added to a final concentration of 1% w/v.

9. The ratio of fluorescein to protein can be estimated by measuring the absorbance at 495 nm and 280 nm. The final F/P ratio should be between 0.3 and 1.0. Lower ratios will yield low signals; higher ratios will give high background.

Calculating the Fluorescein/Protein Molar Ratio

The F/P molar ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate. To determine this ratio, it is necessary to first determine the absorbance of the conjugate sample at 280 nm and then at 495 nm.

Place the conjugate sample in a quartz cuvette. Read the absorbance of the conjugate sample at 280 nm and 495 nm. The absorbance reading of the conjugate sample should be between 0.2 and 1.4 at 280 nm. If the absorbance reading is outside this range, adjust the sample dilution accordingly.

For FITC-IgG conjugates:

For Other FITC-Protein conjugates:

Availability:

Catalog NumberDescriptionSize
100276Fluorescein Isothiocyanate, Isomer I100 mg
250 mg
500 mg
1 g

Also Available:

Catalog NumberDescriptionSize
101713Fluorescein Isothiocyanate On Celite (10%)250 mg
1 g
191368Fluorescein Isothiocyanate-Avidin1 mg
5 mg
10 mg
191294Fluorescein Isothiocyanate- Casein- Agarose5 ml
158062Fluorescein Isothiocyanate-Dextran, Average MW 4000, MW:Number Av. Mol. Wt. not more than 1.5; degree of substitution 0.003-0.020 mole FITC per mole of glucose100 mg
250 mg
158063Fluorescein Isothiocyanate-Dextran, Average MW 20000; degree of substitution 0.003-0.020 mole FITC per mole of glucose100 mg
250 mg
158064Fluorescein Isothiocyanate-Dextran, Average MW 40000, MW:Number Av. Mol. Wt. not more than 1.25; degree of substitution 0.003-0.020 mole FITC per mole of glucose100 mg
250 mg
1 g
158065Fluorescein Isothiocyanate-Dextran, Average MW 70000, MW:Number Av. Mol. Wt. not more than 1.25; degree of substitution 0.003-0.020 mole FITC per mole of glucose100 mg
250 mg
158066Fluorescein Isothiocyanate-Dextran, Average MW 150000, MW:Number Av. Mol. Wt. not more than 1.35; degree of substitution 0.003-0.020 mole FITC per mole of glucose100 mg
250 mg
1 g
191393Fluorescein Isothiocyanate- Streptavidin, supplied in PBS at approximately 0.5 mg/ml0.25 ml
1 ml
797061Protein A- Fluorescein Isothiocyanate1 ml
55881Protein A- Fluorescein Isothiocyanate, supplied in PBS2 mg
622801Protein A- Fluorescein Isothiocyanate5 mg
154095Protein A- Fluorescein Isothiocyanate- Colloidal Gold, 5 nm0.25 ml
0.50 ml
1 ml
191418Dimethyl Sulfoxide, ACS Reagent Grade100 ml
500 ml
1 liter
191406Ammonium Chloride, ACS Reagent Grade500 g
1 kg
5 kg
806801
806803
Xylene Cyanole F.F10 g
25 g
800687
800688
800689
Glycerol, ultra pure500 ml
1 liter
4 liters
195588Sephadex G-75, Globular Proteins 3000-80,000, dextrans 1000-50,00010 g
50 g
100 g
195581Sephadex G-50, fine, globular proteins 1500-30,000, dextrans 500-10,00010 g
50 g
100 g
102891Sodium Azide25 g
100 g
500 g
103700Bovine Serum Albumin1 g
5 g
10 g
25 g
100 g
158394Antibodies: A laboratory manual, by Ed Harlow and David Lane (1988)1 book

FITC conjugated antibodies also available.

References:
  1. Current Protocols in Immunol., Coligan, J.E., et al. (eds), pp. 5.3.3 (Wiley & Sons).
  2. Bridges, C.D. and Fong, S.L., Methods Enzymol., v. 81, 65-77 (1982).
  3. Cherry, N.B., et al., Stain Technol., v. 44, 179 (1965).
  4. Der-Balian, G.P., et al, Anal. Biochem., v. 173, 59 (1988). (FITC labelling of Fab')
  5. Goding, J.W., J. Immunol. Methods, v. 13, 215-226 (1976).
  6. Harlow, E. and Lane, D. (eds), Antibodies: A Laboratory Manual, Cold Spring Harbor, NY, p. 353-355 (1988).
  7. McKinney, R.M., et al., Anal. Biochem., v. 14, 421-428 (1966).
  8. Miller, L., et al., Eur. J. Biochem., v. 174, 23 (1988). (Protein modification of actin at lysine-61.)
  9. Muramoto, K., et al., Anal. Biochem., v. 141, 446 (1984). (Microsequencing of proteins and peptides (HPLC).)
  10. Reisher, J.L. and Orr, H.C., Anal. Biochem., v. 26, 178 (1968). (Removal from sephadex, after filtration of conjugated proteins.).
  11. Rinderknecht, H., Experientia, v. 16, 430 (1960).
  12. Schreiber, A.B. and Haimovich, J., Methods Enzymol., v. 93, 147-155 (1983).
  13. Wilderspin, A.F. and Green, N.M., Anal. Biochem., v. 132, 449 (1983). (Modification of thiol groups.)
  14. Xia, Z., et al., Br. J. Haematol, v. 93, 204-214 (1996).