Eppendorf AF2200 Plate Reader - Manual (Page 61)

Predefined methods
Eppendorf ® PlateReader AF2200
English (EN)
61
6.2.5 Protein quantification BCA, Bradford, Lowry
BCA 562 (600) nm with standards, Bradford 595 (600) nm with standards, Lowry 750 (600) nm with
standards
Introduction
The BCA Protein Assay uses bicinchoninic acid (BCA) for colorimetric quantification of total protein in a
sample. The method is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium. Cu1+
complexes with BCA, forming a colored water-soluble chelate with an absorption maximum at 562 nm.
The Bradford assay is a colorimetric assay based on an absorbance shift of the Coomassie Brilliant Blue
G-250 dye. Under acidic conditions, the green form of the dye is converted into its blue form, which binds
to the protein being assayed. In this process, the absorbance maximum of the dye is shifted from 465 nm to
595 nm.
The Lowry Assay is a colorimetric assay for the quantification of soluble protein. The protein complexes
with cupric sulfate and tartrate in an alkaline solution and reduces the Folin-Ciocalteu reagent. The reaction
product is blue-colored and water-soluble, with an absorption maximum between 500 and 800 nm.
By using two or more protein standards of known concentrations, the protein concentrations of unknown
samples can be obtained.
Default measurement parameters
• Measurement wavelength BCA: 562 (600) (10) nm
• Measurement wavelength Bradford: 595 (600) (10) nm
• Measurement wavelength Lowry: 750 (600) (10) nm
• Number of flashes: 25
• Settle time: 0 ms
• Plate definition: Eppendorf Microplate VIS, 96/F-PS or comparable plates
Using the Eppendorf preconfigured UV/Vis filter slide these methods are measured at 600nm.
As for BCA the absorbance at 600nm is merely half as big as in the BCA absorbance maximum
at 562nm the results of the lower concentrations are slightly inaccurate in comparison to a
measurement at 562nm. The absorbance maxima of Lowry and Bradford are close to 600nm.
Therefore the results of these methods are not restricted by a measurement wavelength of
600nm.
The method is restricted to transparent 96-well microplates.