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Fluorescence methods in life sciences
Ctirad Hofr
Analytical applications of
fluorescence
Determination of concentration
• How to determine quantity of DNA and
protein solution?
• How to determine quality of biomolecules?
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Determination of DNA
concentration
• Using UV absorption spectroscopy it is possible
to determine accurately the concentration down
to the limit 1 µg/ml; the accuracy is about 1%
• The use of fluorescent probe allows to reduce
the detection limit 1000-times - to 1 ng/ml!
• When using fluorescent probes for measuring
the concentration of complex samples (cell
lysate, blood plasma), the effect of the presence
of proteins (absorb at 260nm), nucleotides and
short oligonucleotides is less pronounced.
• The measuring accuracy is about 5%
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Determination of DNA
concentration using Hoechst 33258
• Standard - DNA solution
from 5 – 1000 nM
• Mixed with a solution
Hoechst (200 ng/ml)
in ratio 1:1
• Ex/Em 350/455 nm
• Measured in TE buffer
• Sample consumption ~
50 ng DNA
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Determination of DNA
concentration using PicoGreen
• Detection 25 pg/ml
• Ex/Em 480/520 nm
• Wide dynamic
range – it can
determine the
concentration of
0.025 -1000 ng/ml
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Determination of protein
concentration
• UV absorption - determination based
on the absorption of aromatic AA dependent
on their occurrence in the
sequence (1 mg/ml)
• Bradford method– based on the
change in color after binding to the
protein
(0.25 mg/ml)
• Fluorescent methods (10 ng/ml)
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Fluorescence determination of
protein concentration
• When using fluorescent
probe, NanoOrange is
needed to compensate
differences in protein
structure, which would affect
the interaction - they should
be denatured
• Sensitivity 10ng/ml
• Dynamic range 1000
• Stable for 6 hrs
• Small dependence on the
protein type
• The method is not sensitive
to contamination by other
molecules than lipids
• To quantify in the presence
of lipids or lipoproteins,
CBQCA kit can be used
BSA
A) BSA, trypsin,
carbonic anhydrase
B) IgG, streptavidin,
RNase A
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Determination of the quality and
amount using densitometry of gels
• Simultaneous information about the quality and quantity can
be obtained in the analysis of molecules in the gel
• The amount of absorbed / emitted light is measured
depending on the position of the 2D
• Determination of the concentration of DNA and protein
after gel staining with Coomassie, silver or
fluorescently (SYBR Green, SYPRO Ruby)Light source
Detector