Eppendorf AF2200 Plate Reader - Manual (Page 63)

Predefined methods
Eppendorf ® PlateReader AF2200
English (EN)
63
6.2.6 Protein quantification (NanoOrange 485/595 nm with standards)
Introduction
NanoOrange (Molecular Probes) is a highly sensitive fluorescent stain for protein quantification in solution.
The reagent is virtually non-fluorescent in aqueous solution, but when bound to proteins undergoes a
strong fluorescence enhancement with a broad excitation peak centered at app. 470 nm and a broad
emission peak centered at app. 570 nm.
By using three or more protein standards of known concentrations, the protein concentrations of unknown
samples can be obtained.
Default measurement parameters
• Excitation wavelength: 485 (20) nm
• Emission wavelength: 595 (25) nm
• Number of flashes: 25
• Settle time: 0 ms
• Mode: Top
• Lag time: 0 μs
• Integration: 20 μs
• Gain: "optimal"
• Plate definition: Eppendorf Microplate 96/U-PP, black" or comparable plates
User defined method parameters
• The measurement is performed at 485 / 595 nm with default method parameters.
• Define the number of blank replicates (possible number: 1 – 8).
• Define the number of standards (possible number: 3 – 12).
• Define number of standard replicates (possible number: 1 – 8).
• Define the concentrations of each standard (Press triangle button to open the menu).
• Define the number of samples (max. 92, depends on number of blanks and standards).
• Define the unit of your concentration.
• Check if entered parameters are correct.
• Required plate layout: Pipette in a vertical order, starting with blanks at position A1. Standards are
pipetted in subsequent wells, samples must be placed in the following wells.
• Press Start .
• For method results and data evaluation principle see chapter method results and evaluation procedure .
The method is restricted to 96-well microplates.