The small-scale labeling protocol uses 1 mg IgG at 5 mg/mL in 0.2 mL with a final volume of approximately 0.25 mL. Sephadex G-25M Column A has a maximum sample volume of 0.3 mL, so 1 mL would exceed capacity and may reduce labeling performance. To proceed: concentrate the protein to approximately 5 mg/mL and follow the small-scale method, or if approximately 1 mL is required, switch to Column B with catalog number B4783 (maximum sample volume 1.5 mL) and scale per kit guidance while maintaining the same reagent concentrations and ratios. See the technical bulletin:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/364/319/fitc1bul.pdf
Fortfahren mit
FITC1
FluoroTag™ FITC Conjugation Kit
Synonym(e):
FITC
Anmelden zur Ansicht der Organisations- und Vertragspreise.
Größe auswählen
Ansicht ändern
| Ihnen/SKU | Verfügbarkeit | Preis |
|---|---|---|
1 kit | Warenkorb auf Verfügbarkeit prüfen | € 434,00 |
Über diesen Artikel
NACRES:
NA.32
UNSPSC Code:
12352200
Technischer Dienst
Benötigen Sie Hilfe? Unser Team von erfahrenen Wissenschaftlern ist für Sie da.
Unterstützung erhaltenpackaging
pkg of (Kit contains reagents for 5 conjugations.)
Quality Segment
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.
Biochem/physiol Actions
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.
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
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
1 of 1
Dieser Artikel | |||
|---|---|---|---|
| Quality Level 200 | Quality Level 300 | Quality Level 100 | Quality Level - |
| storage temp. 2-8°C | storage temp. 2-8°C | storage temp. −20°C | storage temp. 2-8°C |
| fluorescence λex 495 nm; λem 525 nm | fluorescence - | fluorescence - | fluorescence - |
| packaging pkg of (Kit contains reagents for 5 conjugations.) | packaging pkg of 1 kit | packaging - | packaging - |
Nur Kit-Komponenten
Produkt-Nr.
Beschreibung
- 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-Komponenten auch einzeln erhältlich
Produkt-Nr.
Beschreibung
SDB & Preisangaben
signalword
Warning
hcodes
Hazard Classifications
Eye Irrit. 2
Lagerklasse
10 - Combustible liquids
flash_point_f
Not applicable
flash_point_c
Not applicable
wgk
WGK 1
Hier finden Sie alle aktuellen Versionen:
Besitzen Sie dieses Produkt bereits?
In der Dokumentenbibliothek finden Sie die Dokumentation zu den Produkten, die Sie kürzlich erworben haben.
-
for small column, can I use 1ml protein at 1mg/ml instead of 0.2ml at 5mg/ml?
1 answer-
Helpful?
-
-
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-
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.
Helpful?
-
-
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.
Helpful?
-
-
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?
-
-
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.
Helpful?
-
-
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?
-
-
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?
-