Analysis Note

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.﹤BR/>﹤BR/>2. Using the two absorbance readings, calculate the F/P molar ratio of the FITC/Protein conjugate using the following equation:﹤div class='Bio_doc_image'>Fig﹤WBR>ure 1: F/P Molar Ration﹤BR/>﹤/div>﹤BR/>﹤BR/>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:﹤div class='Bio_doc_image'>﹤p class='title'>Figure 2: Concentration of FITC-IgG﹤BR/>﹤/div>If the protein conjugated was not IgG, use the following equation to estimate the concentration of conjugated protein:﹤div class='Bio_doc_image'>Fig﹤WBR>ure 3: Concentration of FITC-Protein﹤BR/>﹤/div>Where E﹤SUP>0.1%﹤/SUP> is the A﹤SUB>280﹤/SUB> reading of a 1 mg/ml solution of the unconjugated protein, as measured in a cuvette of 1 cm path length.

Components

Fluorescein (five vials), Carbonate Buffer Concentrate, DMF Solvent, PBS Buffer Concentrate, and a user protocol.

General description

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.

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

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.

Preparation Note

Antibody/Protein Preparation: Add 10 ml of carbonate buffer concentrate to 990 ml of distilled water and dialyze antibody in buffer overnight.

Principle

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.

Warning

Toxicity: Multiple Toxicity Values, refer to MSDS (O)