1. Done by : Rayan Osman
Submitted to Dr. Roland Bou Raad
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Test For Protein Quantification
2. Plan
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Definition of proteins
Ultraviolet-visible spectroscopy
Biuret Method
The Lowry Method
Bicinchoninic Acid Assay (BCA)
Dye-Binding Assays
1. Bradford Assay
3. What is Protein?
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Proteins are complex, specialized molecules
composed of carbon, oxygen, hydrogen, nitrogen and
sometimes sulfur.
The building blocks of proteins are amino acids.
There are 20 different amino acids that combine to
form polypeptides (proteins).
The different amino acids have different side chain,
but are otherwise identical.
Proteins have many important roles in organisms.
Structural proteins such as collagen or elastin,
provide support. Regulatory proteins such as
enzymes control cell processes. Proteins also play an
important part in the immune system (antibodies),
oxygen transport (hemoglobin), movement (muscles)
etc.
4. Ultraviolet-visible spectroscopy
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o A quick, but less specific measure of protein
concentration is the absorbance of the sample at 280
nm. Most proteins have an absorbance maxima at
280 nm as opposed to nucleic acids which absorb at
260 nm. Aromatic residues, such as tryptophan,
tyrosine and phenylalanine are responsible for the
observed absorbance, thus proteins lacking these
residues cannot be measured using this assay.
Requires approximately 50 μg for accurate readings.
o Although nucleic acid contamination can interfere with
measuring protein concentration at 280 nm, a more
accurate measure can be taken by measuring the
absorbance of the sample at both 260 nm and 280
nm and using the following calculation:
o Protein mg/mL = 1.55 A280 – 0.76 A260
5. Advantages:
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Good for samples containing a single protein of
known molar absorptivity
Useful in chromatography to determine elution
profile of protein
Easy, quick assay
Moderate sensitivity
6. Disadvantages:
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Need spectrophotometer capable of reading in
the UV region
Need to use quartz Cuvettes
Cannot use with proteins that do not contain
aromatic residues
Nucleic acid contamination can be a problem
7. Biuret Test
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A test for detecting the presence of peptide
bonds. Under alkaline conditions, Cu2+ forms a
violet-colored complex. Protein concentration is
directly proportional to the intensity of color,
absorbed at 540 nm.
The Biuret reagent contains sodium hydroxide,
hydrated copper (II) sulfate and potassium
sodium tartrate (to stabilize the complexes).
Reduction of copper results in a color change,
which can be read at 550 nm. The linear range is
typically 0.5-20mg protein.
10. Advantages:
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Doesn’t rely on the amino acid composition of the
protein
Does not require a spectrophotometer capable of
measuring in the UV region
Good for whole tissue samples and other sources
of high protein concentration
Relatively few materials interfere with the assay
Simple procedure.
11. Disadvantages:
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Buffers, such as Tris and ammonia, can interfere
with the reaction
Cannot measure the concentration of proteins
precipitated using ammonium sulfate
Low sensitivity – requires higher amounts of
protein
Nucleic acid contamination can be a problem
Proteins with abnormally high or low percentage
of aromatic amino acids will give high or low
readings.
12. The Lowry Method
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The development of the Lowry method (named after
Oliver Lowry) introduced a more sensitive assay for
determining protein concentration. The Lowry method
is sensitive, highly reproducible, inexpensive and
easy to perform.
A modification of the Biuret test, the Lowry method
relies on the reaction of copper with proteins, but the
sample is also incubated with the Folin-Ciocalteu
reagent. Reduction of the Folin-Ciocalteu reagent
under alkaline conditions results in an intense blue
color that absorbs at 750 nm. The Lowry method is
best used with protein concentrations of 0.01–1.0
mg/mL.
Tyrosine, tryptophan, and cysteine of proteins reduce
molybdenum acid and phosphotungstic acid of a
phenol reagent, turning the solution blue.
16. Advantages:
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Easy to use
Highly reproducible
Inexpensive
High Sensitivity and broad linear range
17. Disadvantages:
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Timing and mixing of reagents must be precise
Sensitivity depends on composition of protein as
reaction partly dependent on polar amino acids
Interference from some buffers, particularly
detergents
Complicated procedure with a long preparation.
18. Bicinchoninic Acid Assay (BCA)
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BCA is similar to Lowry, except Bicinchoninic acid
(BCA) is used instead of the Folin-Ciocalteu reagent.
After reduction of Cu2+ ions, two molecules of BCA
chelates with each Cu+ ion resulting in formation of
an intense purple color that absorbs at 560 nm.
BCA is as sensitive as the Lowry method and works
well with protein concentrations from 0.5 μg/mL to 1.5
mg/mL. Although detergents do not interfere as
strongly as in the Lowry method, other contaminants
can interfere with the reaction. In addition, aromatic
amino acids can influence the reaction. However, at
higher temperatures (37-60°C), peptide bonds can
also contribute to formation of the product. Therefore
it is recommended to perform the reaction at higher
temperatures to increase the sensitivity and decrease
the variability due to amino acid composition.
22. Advantages:
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Easy to use
Highly reproducible
Inexpensive
High sensitivity
Simple procedure.
23. Disadvantages:
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Interference from carbohydrates, catecholamines,
tryptophan, lipids, phenol red, cysteine, tyrosine,
impure sucrose or glycerol, uric acid, iron and
hydrogen peroxide
Color continues to develop over time, but is
stable for measurement after 30 minutes at 37°C
24. Dye-Binding Assays
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Dye binding assays rely on an absorbance shift
that occurs when a dye binds to proteins. Several
different dyes can be used: Coomassie Brillant
Blue G-250, bromocresol green, pyrogallol red.
The most commonly used dye-binding assay is
the Bradford Assay.
25. Bradford Assay
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The Bradford assay, is an easy, sensitive and
accurate method for protein quantification.
Binding of Coomassie Brillant Blue G-250 to
proteins, causes a shift of the dye from red (465
nm) to blue (595 nm) under acidic conditions. It is
compatible with more common reagents,
although detergents can cause interference.
Proteins with a concentration of 20-2000 μg/mL
can be measured using the Bradford assay.
29. Advantages:
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Easy to use
Sensitive
Quick
Compatible with many buffers
Very simple operation.
30. Disadvantages:
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Reagent stains cuvettes
Often need to dilute samples prior to analysis
Depends strongly on amino acid composition
Sensitive to detergents although some
companies offer detergent-compatible Bradford
reagents
31. WST
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Reduce WST-8 with proteins with a high pH,
turning the sample blue. Uses the absorption
maximum at 650 nm to determine the quantity