packaging
pkg of (Kit contains reagents for 5 conjugations.)
Quality Level
fluorescence
λex 495 nm; λem 525 nm
storage temp.
2-8°C
Application
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
Analysis Note
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
Kit Components Only
- 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
- P3813Phosphate buffered saline, powder, pH 7.4, for preparing 1 L solutions 5 pkgSDS
related product
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
Certificates of Analysis (COA)
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705578-5MG-PW
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If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
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The lot specific COA document can be found by entering the lot number above under the "Documents" section.
Can Product FITC1, FluoroTag™ FITC Conjugation Kit, be used with antibodies at concentrations less than 5 mg/mL?
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.
Can the columns in Product FITC1, FluoroTag™ FITC Conjugation Kit, be reused?
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.
When using Product FITC1, FluoroTag™ FITC Conjugation Kit, should I adjust the dye:protein ratio for the size of the protein being labeled?
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.
My antibody is in solution containing azide. Can I use the antibody directly in the reaction when using Product FITC1, FluoroTag™ FITC Conjugation Kit?
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.
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