A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)
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 = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon 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 = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. 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.
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis.
M W
MW
Wessendorf
S J
SJ
Tallaksen-Greene
R M
RM
Wohlhueter
D000345:Affinity Labels; D003374:Coumarins / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D008433:Mathematics; D008962:Models, Theoretical; D011506:Proteins / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling
D000345:Affinity Labels; D003374:Coumarins; D005456:Fluorescent Dyes; D011506:Proteins; C051029:7-amino-4-methylcoumarin-3-acetic acid