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Inaccuracy of the Bradford method for the determination of protein concentration in steroid-horseradish peroxidase conjugates

✍ Scribed by Gianfranco Giraudi; Claudio Baggiani; Cristina Giovannoli

Book ID: 104104850
Publisher: Elsevier Science
Year: 1997
Tongue: English
Weight: 443 KB
Volume: 337
Category: Article
ISSN: 0003-2670
DOI: 10.1016/s0003-2670(96)00388-1

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✦ Synopsis

Conjugates between horseradish peroxidase and steroid molecules were prepared with different steroids (progesterone, testosterone, cortisol, estradiol) and different reaction mole ratios. These conjugates were charactetised by liquid chromatographic analysis to check purity grade, stability and the presence of polymeric compounds, and by spectrophotometric measurements to obtain values of the average substitution ratio and of protein concentration by the absorbance corresponding to the Soret band at 402nm. The results obtained showed that the Bradford method, based on interactions between Coomassie Brilliant Blue G-250 and the amine groups on the protein surface, lead generally to an underestimation of protein concentration in steroid-horseradish peroxidase conjugates.

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