Custom Premix Selecting "Custom Premix" option means that all of the beads you have chosen will be premixed in manufacturing before the kit is sent to you.
Provides means to quickly and simply label antibodies or other proteins with fluorescein. Direct labeling of the primary antibody eliminates the need for a secondary antibody, and results in a lower background and overall higher signal-to-noise ratio. Each kit is sufficient to perform at least five conjugations on proteins of 100 µg to 2.5 mg each.
Catalogue Number
343210
Brand Family
Calbiochem®
References
Product Information
Kit contains
Fluorescein (five vials), Carbonate Buffer Concentrate, DMF Solvent, PBS Buffer Concentrate, and a user protocol.
Harmful by inhalation and in contact with skin. Very toxic if swallowed. Contact with acids liberates very toxic gas. Irritating to eyes, respiratory system and skin. May cause sensitization by inhalation and skin contact. May cause harm to the unborn child.
S Phrase
S: 26-36-45-53
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). Avoid exposure - obtain special instructions before use.
Product Usage Statements
Intended use
The Calbiochem® FITC Labeling Kit provides a means of quickly and simply labeling antibodies or other proteins with fluorescein. FITC-antibody conjugates are useful in many applications, such as flow cytometry and immunocytochemistry. Other proteins such as steroids may also be labeled with FITC. Direct labeling of the primary antibody eliminates the need for a secondary antibody, and therefore results in a lower background and an overall higher signal to noise ratio.
Storage and Shipping Information
Ship Code
Ambient Temperature Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
+2°C to +8°C
Protect from Light
Protect from light
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Fluorescein (five vials), Carbonate Buffer Concentrate, DMF Solvent, PBS Buffer Concentrate, and a user protocol.
The Calbiochem® FITC Labeling Kit provides a means of quickly and simply labeling antibodies or other proteins with fluorescein. FITC-antibody conjugates are useful in many applications, such as flow cytometry and immunocytochemistry. Other proteins such as steroids may also be labeled with FITC. Direct labeling of the primary antibody eliminates the need for a secondary antibody, and therefore results in a lower background and an overall higher signal to noise ratio.
Materials provided
This kit contains sufficient reagents to perform 5 conjugations of proteins with 0.1-2.5 mg each.
Antibody/Protein Preparation: Add 10 ml of carbonate buffer concentrate to 990 ml of distilled water and dialyze antibody in buffer overnight.
Detailed protocol
1. Remove the antibody or other protein from dialysis and dilute or concentrate to a final concentration of 2 mg/ml. Place in container suitable for vortexing. 2. Add 850 µl of Solvent Reagent to one vial of FITC. Mix thoroughly. The FITC should now be at a concentration of 1 mg/ml. 3. If the protein being conjugated is not IgG, reserve a sufficient amount of the protein so that a 1 mg/ml solution can be measured in a cuvette of 1.0 cm path length in order to calculate the approximate concentration of the final conjugate (see step 3 of Determining the FITC/Protein Molar Ratio). This reserved protein will be used to calculate E0.1%. 4. For 2.5 mg of antibody or other protein, pipette 500 µl of FITC mixture into the antibody solution and vortex immediately. This provides a conjugation ratio of 200 µg FITC to 1 mg antibody/protein. Mix end-over-end for 2 hours at room temperature, or transfer to a flask and mix with a stir bar and magnetic stirrer. If possible, seal container with aluminum foil while stirring to block out ambient light. 5. Dilute PBS buffer concentrate 1:10 in distilled water (final azide concentration is 0.1%) for dialysis. Reserve sufficient diluted PBS buffer to prepare a spectrophotometric blank for determining the FITC to protein molar ratio. Transfer the conjugate to dialysis tubing and dialyze overnight at room temperature in the diluted PBS. 6. Remove from dialysis tubing. Keep refrigerated (short term) or frozen (long term) and protect from light.
Calculations
1. Read the absorbance of the FITC-antibody conjugate at two wavelengths: 280 nm and 495 nm. Use the 1X PBS/0.1%NaN3 as the spectrophotometric blank. Optimal absorbance readings of the conjugated protein will be within the range of 0.2-1.2 ABS.
2. Using the two absorbance readings, calculate the F/P molar ratio of the FITC/Protein conjugate using the following equation:
Figure 1: F/P Molar Ration
3. There are 2 equations used for the determination of the concentration of the FITC-antibody conjugate. If the protein used for conjugation was IgG, use the following equation to estimate the concentration of the labeled antibody:
Figure 2: Concentration of FITC-IgG
If the protein conjugated was not IgG, use the following equation to estimate the concentration of conjugated protein:
Figure 3: Concentration of FITC-Protein
Where E0.1% is the A280 reading of a 1 mg/ml solution of the unconjugated protein, as measured in a cuvette of 1 cm path length.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
