1.

0 ABSTRACT

This experiment was conducted to learn the principles of protein assays. This exercise
exposed several method of determining protein concentrations. The determination of protein
concentration is an essential technique in all aspects of protein studies and proteomics.Within
this experiment, there are two reagent that involved in the estimation of protein which are
Lowry Reagent and Bradford Reagent.As for Lowry Reagent, there are two materials which
being used in this experiment that is bovine serum albumin (BSA) and gelatin.While, for
Bradford Reagent, the materials that is needed was Coomassie Blue G-250, ethanol and
phosphoric acid.
Initially, started with preparing five different weight of gelatin .Then,they were put in a
tube before dilution process with distilled water in order to become the solution. After 30
minutes, the absorbance value should be determined by using spectrophotometer. The step
also was repeated during the experiment using BSA until the absorbance value is determined
at the end of experiment. According to the theory,the accurate range for the absorbance value
was 0.1 to 1.0.
Secondly, for Bradford Reagent, Coomassie Blue G-250,it must be dissolve in ethanol and
phosphoric acid. The solution that had been mixed together later is be filtered to precipitate
the dye which is blue in colour. For the quantifying of protein, after mix the sample with
Bradford reagent and wait for 5 minutes before measure the absorbance value. At the end of
the experiment, supposedly we need to determine which method is the best following the
criteria given, which are sensitivity of reagent toward protein, convenience, generality and
also linearity.

2.0 INTRODUCTION

Protein assays is most notably quantitation or estimation assays for determining protein
concentration.It is one of the most widely used methods in life science research. Protein
estimation of protein concentration is necessary in protein purification, electrophoresis, cell
biology, molecular biology, and other research applications. There are soem unique selection
of protein estimation assays that are improvements on the Biuret, Lowry, BCA and Bradford
assays.

Lowry Assay
The Lowry assay is based on the Biuret reaction of proteins with cupric sulfate at alkaline
conditions and theFolin-Ciocalteau phosphomolybdotungstate reduction.The use of FolinCiocalteau reagent greatly enhances the sensitivity of the assay (down to 10 ug/ml).The assay
is pH dependent (optimum at pH 10 to 10.5),and thus it is important to maintain the pH
during the assay.Color reaction is stable for several hours, and thus the reactions can be
measured at any time after 10 min.But the Folin reagent is not very stable in alkaline
condition and only reactive for the first few minutes after addition, and thus the mixing is
critical to obtain reproducible results.
In this assay, color develops in two steps. The peptide bonds of proteins first react with cupric
sulfate under alkaline condition, producing Cu+, and the subsequent reduction of Folin
reagent (phosphomolybdotungstate) by the copper-treated protein to heteropolymolybdenum
blue.Color develops in the second reaction (i.e., the reaction with phosphomolybdotungstate)
and is primarily due to the amino acids tyrosine and tryptophan, and to a lesser extent cystine,
cysteine, and histidine.The blue color has the maximum absorbance at 750 nm.

Image source from google :reaction of cupric sulphate with peptide bond
Bradford Assay
The Bradford protein assay has become the preferred method for many investigators, because
it is simple and rapid compared to the Lowry method.Moreover, this assay is comparatively
free from interference by common reagents except detergents.The assay involves the use of
Coomassie Brilliant Blue G-250, which reacts primarily to basic (especially arginine)and
aromatic amino acids.The Bradford protein assay is performed in two formats:standard assay
(0.1 to 1 mg/ml) and a microassay (5 to 40g/ml) for use with a microplate reader.
The assay is based on the immediate absorbance shift 470 nm to 595 nm that occurs when
dye binds to protein in acidic solution.The dye is believed to bind to protein via electrostatic
attraction of the dye's sulfonic acid groups (Figure 2.6 A).The mechanism of dye binding can

be explained by the dye existing as three absorbing species, a red cationic species (Amax 470
nm), a green neutral species (Amax 650 nm), and a blue anionic species (Amax 595 nm).

-Image source from Google:structures of commasie blue

Bovine serum albumin (BSA) is commonly used in cell culture protocols, particularly
where protein supplementation is necessary and the other components of serum are
unwanted. In cell culture, its main role is as a carrier of small molecules. Because of its
negative charge, Bovine Serum Albumin binds water, salts, fatty acids, vitamins and
hormones, then carries these bound components between tissues and cells. The binding
capacity also makes Bovine Serum Albumin an effective scavenger to remove toxic
substances, including pyrogens, from the medium.Albumins are readily soluble in water and
can only be precipitated by high concentrations of neutral salts such as ammonium sulfate.
The solution stability of Bovine Serum Albumin is very good (especially if the solutions are
stored as frozen aliquots). Albumins are frequently used as stabilizers for other solubilized
proteins (e.g., labile enzymes). However, albumin is readily coagulated by heat. When heated
to 50C or above, albumin quite rapidly forms hydrophobic aggregates which do not revert
to monomers upon cooling. At somewhat lower temperatures aggregation is also expected to
occur, but at relatively slower rates.Albumin is used to solubilize lipids, and is also used as a
blocking agent in Western Blot or ELISA applications
http://www.biowest.net/products/serum/bovine-serum-albumin-bsa/-[1]
Gelatine contains specific amounts of 18 different amino acids (AA) which are joined
together in sequences to form polypeptide chains of ca. 1000 AA per chain, scientifically
known as the primary structure. Three of the polypeptide chains formed this way join
together as a left-hand spiral to give the secondary structure. In the tertiary structure, the
spiral winds and folds itself to a right-hand spiral (triple helix). This results in a rod-shaped
molecule, the so-called proto fibril.[2]

3.0 OBJECTIVES

At the end of experiment,student should be able -:

i.
ii.
iii.
iv.
v.

To learn the principles of protein assays.

To determine the concentration of proteins by Lowrys method.
To determine protein concentrations using the Bradford Assay.
To understand the concept of a colorimetric assay and its limitations.
To become familiar with the Beers Law and use of a spectrophotometer for analytical
experiments.

4.0 THEORY

As been stated earlier,we only used Bradford and Lowry methods in order to determine
protein concentrations in our experiment.Due to some problem,Biuret assay cannot be
conducted. Each colometric assay has its limitations and advantages.The Bradford assay uses
the common Coomassie Brilliant Blue dye, which makes it convenient. However, it is
sensitive to the amino acid composition of the protein and a plot of absorbance vs.
concentration using it is only linear over a short range of protein concentrations.Although, a
majority of biological and biochemical laboratories has at least at some point applied one of
these methods, the detection and determination of some protein concentrations might not be
satisfactory, if possible at all.
Spectrophotometry has a wide range of applications. This technique can be used to quantify
the amount of DNA, RNA, or protein in a sample, the concentration of bacterial cells in a
culture, or the concentration of pigments in a solution, as well as many others. Absorbance
values are obtained by determining the amount of light of a certain wavelength that is
absorbed by a sample. Beers Law is used to relate the absorbance for a sample to its
concentration, the equation is:
A = lc;
Whereby -:
is the molar extinction coefficient of the material,
l is the path length the light must travel through the sample (usually 1 cm) and
c is the concentration of the sample solution.
In proteins, it is often better to use E1% values as opposed to the molar extinction coefficient.
This is due to the fact that is using the absorbance of a 1M solution, and E1% is using the
absorbance of a 1% w/v solution. Many times when dealing with proteins the MW is very
large, and creating a 1M solution is quite difficult; however, a 1% w/v solution is relatively
simple. To convert E1% values to the molar extinction coefficient, the following equation can

be used: = (E1%*MW)/10. In this lab you will examine the interactions of light with bovine
serum albumin (BSA), a common protein, using a spectrophotometer to measure the
concentration of BSA in an unknown sample. Beers law will be used to perform this
task.The direct relationship between absorbance and concentration for a solution is known as
Beer's law.