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Related Experiment Videos

The variable reagent blank: protein determination as a model.

D Bozimowski, J D Artiss, B Zak

    Journal of Clinical Chemistry and Clinical Biochemistry. Zeitschrift Fur Klinische Chemie Und Klinische Biochemie
    |October 1, 1985
    PubMed
    Summary
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    This study compares three protein assays, finding that Biuret and Coomassie Brilliant Blue methods show significant reagent blank deviations due to overlapping spectra. The Folin-Ciocalteau assay, however, remains unaffected, serving as a reliable control.

    Area of Science:

    • Biochemistry
    • Analytical Chemistry

    Background:

    • Accurate protein quantification is crucial in biochemical analysis.
    • Reagent blank effects can introduce significant errors in spectrophotometric assays.
    • Understanding these effects is vital for assay validation.

    Purpose of the Study:

    • To analyze deviations in measured reagent blanks versus true reagent blanks in three total protein assays.
    • To investigate the impact of overlapping spectra on assay accuracy.
    • To identify a reliable assay for controlling blank deviations.

    Main Methods:

    • Comparative analysis of three total protein assays: Biuret, Coomassie Brilliant Blue, and Folin-Ciocalteau.
    • Evaluation of reagent blank behavior under varying protein concentrations.
    • Spectrophotometric analysis at the assay wavelength.

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    Main Results:

    • Biuret and Coomassie Brilliant Blue assays exhibited pronounced deviations in true reagent blanks.
    • The Folin-Ciocalteau reaction showed no deviation as its blank did not absorb at the assay wavelength.
    • Overlapping spectra between reagent and protein complex caused the blank effect in Biuret and Coomassie assays.

    Conclusions:

    • The Folin-Ciocalteau assay serves as a suitable reference for assessing reagent blank deviations.
    • Biuret and Coomassie Brilliant Blue assays are susceptible to significant errors from variable reagent blanks.
    • Assay selection should consider spectral properties to minimize blank-related inaccuracies.