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Alyssa Musni, Martin Louie Naraja, 
 
, Adreinne
Nazaire, Krystal Anne C. New
Group 5 2E Pharmacy BioChemistry Laboratory


The Bradford protein assay is a spectroscopic analytical procedure used to
measure the concentration of protein in a solution. This experiment aims to
determine the concentration of the unknown protein solution and to draw
the standard curve by plotting the 595nm (A595) against a reagent blank.
Standards were prepared by adding 0.3 and 0.4mL of BSA stock solution.
Distilled water was added to each of the test tube to bring the volume to
1mL. For the determination of the unknown concentration, 1mL of the
unknown protein sample was used. Through the use of the
spectrophotometer, the absorbances (in nm) for the unknown proteins were
determined. A standard curve was drawn by plotting the A595 versus the BSA
concentration. The concentrations of the unknown proteins were solved by
using linear regression.
j

Protein
Bradford Assay Method
Spectrophotometer
BSA standard (bovine serum albumin)

ÿÿè The most commonly used

There is no single protein assay assays are the Ultraviolet
method that yields absolutely Absorbance, Lowry Assay, BCA
accurate results. The only true and assay and the Bradford Assay. The
accurate method for determining UV absorbance monitors the
protein concentration is by acid absorbance of aromatic amino
hydrolyzing a portion of the sample acids, tyrosine and tryptophan or if
and then carries out amino acid the wavelength is lowered, the
analysis on the hydrolyzate. But, absorbance of the peptide bond. It
this method is time-consuming. is quick, with the samples that can
Each method and assay has its be recovered. But, it is also highly
own disadvantage and limitations. susceptible to contamination by
buffers, biological materials and
salts. The Lowry Assay or
enhanced copper since it reduces useful protein concentration
Cu+2 to Cu+1 , sensitive over a wide range. The response is also
range and is the most commonly protein dependent, and varies with
referenced procedure for protein the composition of the protein.
determination but, it also takes a These limitations make protein
considerable amount of time. And standard solutions necessary.
the BCA assay or the bicinchoninic
acid which is less susceptible to A spectrophotometer is
interference from common buffer employed to measure the amount
substance and is very sensitive and of light that a sample absorbs. The
rapid if you use elevated instrument operates by passing a
temperatures, but, the reaction beam through a sample and
does not go to completion when measuring the intensity of light
performed at room temperature reaching a detector. The beam of
and dilution is often necessary for light consists of a stream of
concentrated protein samples. photons. When, a photon
encounters an analyte molecule,
The Bradford protein assay is a there is a chance the analyte will
spectroscopic analytical procedure absorb the photon. This absorption
used to measure the concentration reduces the number of photon in
of protein in a solution. It is the beam of light, thereby reducing
dependent on the amino acid the intensity of the light beam.
composition of the measured
protein. It is more efficient than The objective of this
other methods because assay it is experiment is to determine the
faster, involves fewer mixing steps, concentration of the unknown
does not require heating, and gives protein solution and to draw the
a more stable colorimetric standard curve by plotting the 595
response than other methods. nm (A595) against a reagent blank.
p
The Bradford assay is faster, cÿ
involves fewer mixing steps, does The materials used in this
not require heating, and gives a experiment are protein isolate,
more stable colorimetric response. Bradford reagent, and Bovine
Its response is prone to influence SerumAlbumin Standard. A
from non protein sources and spectrophotometer was used to
becomes progressively more measure the absorbance of the
nonlinear at the high end of its proteinsolution. A vortex was also
utilized to facilitate the mixing of Bradford reagent was likewise
the reagent to the protein prepared. Distilled water was
solutions.Each member of added to eachtube to bring the
the group was given a protein volume to 1 mL. 10 mL of Bradford
isolate with a concentration that is reagent was also added to
unique to theother members of the each tube. 0.10mL and 0.20 mL of
group.The Bradford Reagent was the unknown protein solution was
prepared by dissolving 100 mg of used and was then added with
Coomassie Blue G250 in 50 mL distilledw a t e r to bring its
of 95% ethanol. Then, the solution total v olum e to 1 mL.
was mixed to 100 mL of 85% Using the previously
phosphoric acid and made up to 1L prepared reagent bl ank,
by addi ng distilled water. t h e spectrophotometer was
The reagent was filtered calibrated to zero. The absorbance
and stored i n an amber of the standards and
b o t t l e a t r o o m temperature.The the unknownprotein solution was
protein standard used in this read at 595 nm against the
experiment was bovine serum reagent blank after 5 minutes but
albumin. Bovine serum albuminis a before 1 hour.The standard curve
globular protein obtained from was made by plotting the
cattle. It is frequently used as a concentration of the BSA standards
standard because it is low-priced against theabsorbance read (A
and is easily available in pure form. 595). The concentration of the
Also, because many studies have protein solution in reference to the
previously usedbovine serum standardcurve for bovine serum
albumin as its protein standard, albumin was calculated.
the results of the assay can be
directly comparedwith the results
in these aforementioned studies.
The amount of the protein standard
used in thisexperiment is 0.20
mg/mL in water.A set of standards
containing 0.20, 0.30, 0.40, 0.50,
0.60, 0.80, and 1.0 mL of bovine
serumalbumin stock solution was
prepared. A reagent blank 
consisting of 1.5 mL of distilled 
water mixed with 7.5 mL of