YouTube Transcript:
How to determine the Protein Concentration with the Bradford Assay

the bread for test or bradford assay
is a technique used for protein
quantification
it is one among many colorimetric assays
used to determine the total protein
concentration
in a solution the bradford method is
based on a dye
named komasi brilliant blue in an acidic
solution
this reagent is protonated and has a
reddish or brownish color
in the presence of protein komasi
brilliant blue stably binds to them
the dye protein complex quickly takes a
blue color
required for this reaction is an acidic
environment the complex formation
results in a shifted absorption maximum
of the dye
the protein solution can be measured in
a spectrophotometer
at 595 nanometers
in the absence of protein there is no
change in color
the absorption maximum does not shift
how does komasi brilliant blue g250
interact with the proteins this
is kumasi brilliant blue in an acidic
environment
in the presence of protein the reagent
disrupts the protein structure
that exposes the amino acid side chains
the sulfonic acid groups of the dye
interact with the positive amino groups
of the protein
found in basic amino acid residues
present in lysine
arginine and histidine this results in a
stable form of the dye
and the color turns blue after dye
protein complex formation
but how do we determine the protein
concentration
the spectrophotometer measures the
optical density
at 595 nanometers
first the pure solution without any
protein content is measured
to calculate the protein concentration
of an unknown sample
we need a reference solution where the
protein concentration
is already known this is classified
as the standard in most cases the
protein
bsa is used for this different dilutions
of this standard are made
the optical density is measured for 1
microgram per microliter
and higher concentrations such as 2
and 4 microgram per microliter that
generates a linear curve
in which the optical density increases
proportionally
with protein concentration now
the sample with the unknown protein
content is measured
the spectrophotometer assigns the
optical density
and with extrapolation we can calculate
the protein concentration which will be
3 microgram
per microliter the bread for test has
advantages
and disadvantages one advantage
is that the komasi dye interacts quickly
with the proteins
the assay can determine protein
concentrations
in a very short time it is also
easy to use since it just requires the
bradford reagent
and a spectrophotometer the assay can be
performed at room temperature
in comparison to other protein
quantification techniques
the bradford method is extremely
sensitive
it can even detect protein
concentrations of
one microgram per milliliter one of the
disadvantages
is interference with detergents such as
sds since the commercial dye
reacts mainly with the basic amino acids
the assay
faces another limitation it is dependent
on the protein's amino acid composition
the bradford assay is just one among
many protein quantification techniques
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