Conjugation with FITC occurs through free amino groups of proteins or peptides. The F/P molar ratio of the conjugate, as well as the limit of detection, will vary from one protein to another and must be determined empirically by the end user. Please access the Product Information Sheet in the DOCUMENTATION section of the Product Detail Page under the 'Product Information Sheet' tab for further details.
FITC1
FluoroTag™ FITC Conjugation Kit
Synonym(s):
FITC
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About This Item
UNSPSC Code:
12352200
NACRES:
NA.32
Pricing and availability is not currently available.
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packaging
pkg of (Kit contains reagents for 5 conjugations.)
Quality Level
fluorescence
λex 495 nm; λem 525 nm
storage temp.
2-8°C
Application
The Flourotag FITC Conjugation Kit can be used for Immunohistochemisty and Immunocytochemistry using Flow Cytometry. It is also be used for the conjugation of FITC to peptide hormones, cytokines, growth factors and proteins.
Features and Benefits
- Suitable for both small (1 mg) and large (5 mg) scale conjugations
- Completely aqueous procedure - no DMF needed
- Fast gel filtration separation of conjugate from excess FITC
- Complete protocols for conjugation and F/P ratio determination
- Sufficient reagents for at least 5 conjugations of 5 mg protein each and for optimization of F/P ratio before scale-up
- References for applications and protocols
Principle
Sigma offers a convenient kit for preparing FITC-labeled antibodies. Fluorescein isothiocyanate (FITC), Isomer 1, is a widely used fluorophore, popular because of its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through free amino groups of proteins or peptides, forming a stable thiourea bond (see reaction). FITC conjugates of antibodies, lectins, hormones, and growth factors have been used in a variety of immunohistochemical and flow cytometry applications. The protocols have been optimized for antibodies, but may be adapted to other proteins by the end user.
Analysis Note
Procedure
1. Dissolve protein and FITC in carbonate-bicarbonate buffer.
2. Slowly add FITC to protein with stirring. Cover with foil and stir 2 hours at room temperature.
3. Separate conjugate from free FITC on G-25 column. Collect fractions.
4. Pool fractions containing conjugate.
5. Determine F/P ratio of conjugate spectrophotometrically.
6. Stabilize with 1% bovine serum albumin and 0.1% sodium azide and store at 0-5 °C.
1. Dissolve protein and FITC in carbonate-bicarbonate buffer.
2. Slowly add FITC to protein with stirring. Cover with foil and stir 2 hours at room temperature.
3. Separate conjugate from free FITC on G-25 column. Collect fractions.
4. Pool fractions containing conjugate.
5. Determine F/P ratio of conjugate spectrophotometrically.
6. Stabilize with 1% bovine serum albumin and 0.1% sodium azide and store at 0-5 °C.
Legal Information
FluoroTag is a trademark of Sigma-Aldrich Co. LLC
Kit Components Only
Product No.
Description
- Sephadex G-25 column, 3.5 mL 1
- Sephadex G-25 column, 9.1 mL 1
- Fluorescein isothiocyanate isomer I - F7250KC-2MG 2 mg
Kit Components Also Available Separately
Product No.
Description
SDS
- P3813Phosphate buffered saline, powder, pH 7.4, for preparing 1 L solutions 5 pkgSDS
related product
Product No.
Description
Pricing
Signal Word
Warning
Hazard Statements
Precautionary Statements
Hazard Classifications
Eye Irrit. 2
Storage Class Code
10 - Combustible liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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I would like to inquire about the sensitivity, limit of detection (LOD), and limit of quantification (LOQ) for the FluoroTag™ FITC Conjugation Kit. Thank you.
1 answer-
Helpful?
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Can Product FITC1, FluoroTag™ FITC Conjugation Kit, be used with antibodies at concentrations less than 5 mg/mL?
1 answer-
You can scale down the reaction for labeling. A possible problem occurs in using the column to remove the free FITC. If the volume is too small, the column cannot be used. We would recommend using dialysis to remove the free (unconjugated) FITC.
Helpful?
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My antibody is in solution containing azide. Can I use the antibody directly in the reaction when using Product FITC1, FluoroTag™ FITC Conjugation Kit?
1 answer-
It is not recommended to have azide in the reaction mix. If the buffer contains amines or sodium azide, dialyze protein solution (1 mL) against PBS, pH 7.4 (approximately 1000 mL), overnight at 2-8 °C.
Helpful?
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When using Product FITC1, FluoroTag™ FITC Conjugation Kit, should I adjust the dye:protein ratio for the size of the protein being labeled?
1 answer-
Adjustments for dye/protein ratios should only be made for very small proteins, i.e. < 25 kDa. Here, you should use less protein per dye portion. In many cases, this weight amount of protein is IgG with MW 150 kDa, and the kit would provide a molar excess of dye to provide attachment of a few dyes per IgG. If one wants to attempt labeling of a very small protein using the same weight ratio, there is a good chance there would not be enough dye/protein.
Helpful?
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Can the columns in Product FITC1, FluoroTag™ FITC Conjugation Kit, be reused?
1 answer-
Wash the column with 35 ml (10 X column volumes) of PBS to remove unbound fluorophore. This is sufficient to regenerate the column. For prolonged storage, wash the column with 10 mL of PBS containing 0.05% sodium azide and store capped at 2-8 °C, with about 1 mL buffer above the gel.
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What is the Department of Transportation shipping information for this product?
1 answer-
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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