## Abstract

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = ((ε_{350}(AMCA)) · A_{280} - (ε_{280}(AMCA)) · A_{350}) ((̇_{280}(IgG)) · A_{350} - (̇_{350}(IgG)) · A_{280}) where A is the optical density of the conjugate at the given wavelength and ε is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10^{4} at 350 nm and 8.29 x 10^{3} at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10^{3} at 350 nm and 1.26 x 10^{5} at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = ((1.90 x 10^{4}) · A_{280} - (8.29 x 10^{3}) · A_{350}) ((1.26 x 10^{5}) · A_{350} - (1.56 x 10^{3}) · A_{280}). Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.

Original language | English (US) |
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Pages (from-to) | 87-94 |

Number of pages | 8 |

Journal | Journal of Histochemistry and Cytochemistry |

Volume | 38 |

Issue number | 1 |

DOIs | |

State | Published - 1990 |

## Keywords

- AMCA
- aminomethylcoumarin acetic acid
- f/p ratio
- immunocytochemistry
- immunofluorescence