Practical no. :
2
Experiment Title
: Protein Experiment
Objectives :
1. Learn
the principles of protein assays.
2. Determine
protein concentrations using the Biuret Protein Assay.
3. Determine
protein concentrations using the Lowry Assay.
a) Biuret
assay
Abstract
:
This
experiment is conducted to learn the principles of protein assays. This
exercise introduces students to method of determining protein concentrations. The
determination of protein concentration is an essential technique in all aspects
of protein studies and proteomics. This lab activity is designed to teach
students the principles behind a common protein estimation assay known as the
Biuret Protein Assay (absorbance at 540nm).
Standard Biuret reagent is already
prepared. The 0.50mL of protein is mixed with the prepared Biuret Reagent. The
solution is mixed well and allows standing for 10 minute. The absorbance for
each tube is read against the blank at 540 nm. The standard curve is plotted
using concentration of standard (mg/mL) against the absorbance at 540 nm. The
standard is used to determine the concentration of the protein in the five egg
samples from regular chicken, quail, duck, and omega. To analyze the data, a
line or curve is fit through the standards. For the sample, read across the
graph from the spot on the Y-axis that corresponds to absorbance of the sample
until the standard curve is intersected. Read down the graph until intersect the
X-axis. The concentration of protein in the sample is the value on the X-axis.
Introduction
:
The Biuret reaction can be used for both qualitative and quantitative analysis of
protein. The biuret method depends on the presence of peptides bonds in
proteins. When a solution of proteins is treated with cupric ions (Cu2+)
in a moderately alkaline medium, a purple colored Cu2+- peptide
complex is formed which can be measured quantitatively by spectrophotometer in
the visible region. So, biuret reagent is alkaline copper sulfate solution.
The
intensity of the color produced is proportional to the number of peptide bonds
that are reacting, and therefore to the number of protein molecules present in
the reaction system. The reaction do not occur with amino acids because the
absence of peptide bonds, and also that with di-peptide because presence of
only one peptide bond, but do with tri-, oligo-, and poly-peptides. Biuret
reaction needs presence of at least two peptide bonds in a molecule.The
reaction occurs with any compound containing at least two bonds of:
The
reaction takes its name "Biuret Reaction" from the fact that biuret
itself, obtained by heating urea, gives a similar colored complex with cupric
ions.
Apparatus and Materials
:
Protein
samples
Standard
solutions
Distilled
water
Biuret
reagent
Test
tubes
Label
Test
tube rack
Pipettes
Methods
:
Result:
Protein’s
content:
Ayam
biasa :
4.55mg/ml x 50x dilution = 227.5 mg/ml
Ayam
kampung :
4.70mg/ml x 50x dilution = 235.0 mg/ml
Ayam
omega :
3.40mg/ml x 50x dilution = 170.0 mg/ml
Puyuh :
5.40mg/ml x 50x dilution = 270.0 mg/ml
Itik :
4.79mg/ml x 50x dilution = 239.5 mg/ml
Discussions:
In this experiment, we have to investigate about the protein
contents (concentration) in the five egg’s samples which are telur ayam
biasa, ayam kampung, ayam omega, puyuh and itik. We used Biuret reagent
test to test the content of protein in all those egg samples. The Biuret test
is a chemical test used for detecting the presence of peptide bonds. In the
presence of peptides, a copper(II) ion forms violet-colour coordination complexes
in an alkaline solution. The Biuret reaction can be used to assess the
concentration of proteins because peptide bonds occur with the same frequency
per amino acid in the peptide. The intensity of the colour and hence the
absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.
Before we tested the sample, we have plot the standard curve. After
that, we have to dilute the egg samples 50x because if we do not dilute those
sample, we cannot get the amount of the protein content. This is because the
amount of protein have exceed the standard scale. From our results, we found
out that quail egg have highest content of protein followed by duck egg, ayam
kampung, ayam biasa and lastly ayam omega. The amount of protein
contents in those eggs were 270.0 mg/ml, 239.5 mg/ml, 235.0 mg/ml, 227.5 mg/ml and 170.0 mg/ml.
As we have known before, omega egg should have higher content of
nutrition. But it actually have higher amount of fatty acid. This is because sources
of omega-3 fatty acids have been mixed in with the chicken feed but their
protein contents was the lowest compared to the other eggs.
The new information about the omega-3 egg, egg whites contain more
protein than egg yolks and they also have less fat. The egg white from one
large omega-3-enriched egg contains 3.6 g of protein, while the egg yolk
contains only 2.7 g. Most of an egg's fat is in the yolk. The yolk from one
large omega-3-enriched egg contains 4.51 g of fat, including 1.6 g of saturated
fat. The unsaturated fat in these yolks provides a good source of omega-3 fatty
acids. The egg white, on the other hand, contains only 0.06 g of fat and no
saturated fat. The yolk contains 210 mg of cholesterol, while the white
contains no cholesterol.
References:
Biuret
test. Retrieved on April 1, 2013 from http://en.wikipedia.org/wiki/Biuret_test
Lisa Porter (2011). Egg protein levels. Retrieved on April
1, 2013 from http://www.livestrong.com/article/470871-egg-protein-levels/#ixzz2PH9KhV7I
Cavemangreg (2011). All about eggs. Retrieved on April 1,
2013 from http://www.paleodietandliving.com/paleo-diet/all-about-eggs/
Title:
Lowry Assay
Abstract :
The
determination of protein concentration is an essential technique in all aspects
of protein studies and proteomics. This lab activity is designed to teach
students the principles behind a common protein estimation assay known as Lowry
Protein Assay. Although there are a wide variety of
protein assays available, none of the assays can be used without first
considering their suitability for the application. Each method has its own advantages and
limitations and often it is necessary to obtain more than one type of protein
assay for research applications. Protein assays based on these methods are
divided into two categories, dye binding protein assays and protein assays
based on alkaline copper. Under alkaline conditions cupric ions (Cu2+)
chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu). The Cuprous ions
can also be detected with Folin Ciocalteu Reagent (phosphomolybdic/phosphotungstic
acid) and this method is commonly referred to as the Lowry method. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a blue color
that can be read at 750nm. The amount of
color produced is proportional to the amount of peptide bonds, i.e. size as
well as the amount of protein/peptide. An experiment on Lowry Assay is
conducted to learn the principles of protein assays and to determine the
protein concentration using Lowry Protein Assay.
Introduction:
The “Lowry Assay:
Protein by Folin Reaction” (Lowry et al.,
1951) has been most widely used method to estimate the amount of proteins
(which is already in solution or easily-soluble in dilute alkali) in biological
samples. First the proteins are pre-treated with copper ion in alkali solution,
and then the aromatic amino acids in the treated sample reduce the
phosphomolybdatephosphotungstic acid present in the Folin Reagent. The end
product of this reaction produced a blue color solution. The amount of proteins
in the sample can be estimated via reading the absorbance at 750nm of the end
product of the Folin reaction against a standard curve of a selected standard
protein solution and sample protein solution. Lowry's
assay is not without problems. In particular, it is sensitive to
"interference" by many other
compounds. Interference, the production of color by substances other that the
analyte of interest, is a common problem with
indirect colorimetric assays. In an attempt to overcome
some of the problems of Lowry's method, many other assays for protein have been
proposed. Two other colorimetric methods commonly
used for the assay of protein are the "Bradford"
assay and the "BCA assay". The Bradford" is based on a shift in
the spectrum of a dye upon binding to
proteins. The "BCA assay" is based upon the detection of Cu(I) (produced when proteins react with alkaline Cu(II))
using bicinchoninic acid (BCA).
Materials and apparatus:
Solutions
of standard protein
Solutions
of sample protein (telur ayam biasa, telur puyuh, telur itik, telur ayam
kampung, telur ayam omega)
Lowry
reagents 1 and 2
Distilled
water
Test
tubes
Test
tube rack
Absorbance machine
Absorbance machine
Methods
Discussion
The Lowry method is one of the most sensitive and
widely used. The Lowry procedure can detect protein levels as low as 5 µg. There
are several limitations to this method. Several contaminants interfere with the
assay. With most protein assays, sample protein concentrations are determined
by comparing their assay responses to that of a dilution-series of standards
whose concentrations are known. Protein samples and standards are processed in
the same manner by mixing them with assay reagent and using a spectrophotometer
to measure the absorbance. The responses of the standards are used to plot or
calculate a standard curve. Absorbance values of unknown samples are then
interpolated onto the plot or formula for the standard curve to determine their
concentrations.
Standard
Curve
This comparative method for determining the
concentration of an "unknown" is conceptually simple and
straightforward. However, its implementation in an assay protocol is
complicated by pipetting and dilution steps, evaluation of replicates,
blank-corrections and other factors. These steps frequently cause confusion
with regard to the calculations that are necessary to obtain a final
determination.
The Lowry method
relies on two different reactions. The first is the formation of a copper ion
complex with amide bonds, forming reduced copper in alkaline solutions. This is
called a "Biuret" chromophore. The second is the reduction of
Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and
tryptophan residues. The reduced Folin-Ciocalteu reagent is blue and thus
detectable with a spectrophotometer in the range of 500-750 nm. The Biuret
reaction itself is not all that sensitive. Using the Folin-Ciocalteu reagent to
detect reduced copper makes the assay nearly 100 times more sensitive than the
Biuret reaction alone.
The assay is
relatively sensitive, but takes more time than other assays and is susceptible
to many interfering compounds. The following substances are known to interfere
with the Lowry assay: detergents, carbohydrates, glycerol, Tricine, EDTA, Tris,
potassium compounds, sulfhydryl compounds, disulfide compounds, magnesium and
calcium. Most of these interfering substances are commonly used in buffers for
preparing proteins. This is one of the major limitations of the assay. The
Lowry assay is sensitive to variations in the content of tyrosine and tryptophan
residues. If the protein we are assaying has an unusual content of these
residues, an appropriate substitute standard is required. The standard curve is
linear in the 1 to 100 ug protein region. The absorbance can be read in the
region of 500 to 750 nm. Most researchers use 660 nm, but other wavelengths
also work and may reduce the effects of contamination (e.g. chlorophyll in
plant samples interferes at 660 nm, but not at 750 nm).
The Lowry protein
assay offered a significant improvement over previous protein assays. The
Modified Lowry Protein Assay uses a stable reagent that replaces two unstable
reagents described by Lowry. Essentially, the assay is an enhanced biuret assay
involving copper chelation chemistry. Although the mechanism of color formation
for the Lowry assay is similar to that of the BCA protein assay, there are
several significant differences between the two. The exact mechanism of color
formation in the Lowry assay remains poorly understood. The assay is performed
in two distinct steps.
First, protein is
reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes
at room temperature. During this incubation, a tetradentate copper complex
forms from four peptide bonds and one atom of copper (this is the "biuret
reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is
added. This compound (called Folin-phenol reagent) becomes reduced, producing
an intense blue color. It is believed that the color enhancement occurs when
the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic
acid complex. The blue color continues to intensify during a 30 minute room
temperature incubation. It has been suggested that during the 30 minute
incubation, a rearrangement of the initial unstable blue complex leads to the
stable final blue colored complex which has higher absorbance. The final blue
color is optimally measured at 750nm, but it can be measured at any wavelength
between 650nm and 750nm with little loss of color intensity. It is best to
measure the color at 750nm since few other substances absorb light at that
wavelength.
For small peptides,
the amount of color increases with the size of the peptide. The presence of any
of five amino acid residues (tyrosine, tryptophan, cysteine, histidine and
asparagine) in the peptide or protein backbone further enhances the amount of
color produced because they contribute additional reducing equivalents to
further reduce the phosphomolybdic/phosphotungstic acid complex. With the
exception of tyrosine and tryptophan, free amino acids will not produce a
colored product with the Lowry reagent, however, most dipeptides can be
detected. In the absence of any of the five amino acids listed above in the
peptide backbone, proteins containing proline residues have a lower color
response with the Lowry reagent due to the amino acid interfering with complex
formation.
There are several
ways to measure protein concentration, and each of them has its own advantages
and disadvantages. The three different methods for measuring protein concentration
are absorbance at 280 nm, the Bradford assay, and the BCA assay.
Absorbance at 280 nm
How it works:
Aromatic residues,
like tyrosine and tryptophan, absorb UV light at 280 nm. So, if we have an extinction coefficient for
the protein , we can measure the
absorbance in a UV/Vis spectrometer and calculate the concentration of your
protein using Beer’s law (A = elc, where l is the path length of the
spectrometer). We can estimate the
extinction coefficient of the protein based on the sequence using Expasy’s
ProtParam tool. Because ProtParam only
considers the linear sequence of the protein and doesn’t take into account the
structure, which can affect the extinction coefficient, we’ll want to denature
the protein before we measure the absorbance.
Advantages:
This technique is
quick and doesn’t require any special reagents, except for the guanidinium,
which you may have on hand anyway.
Disadvantages:
This method relies
on having an accurate extinction coefficient for the protein, which depends on
the number of aromatic residues. If
there aren’t a decent number of aromatic residues, our extinction coefficient
will be quite low, and we will need a fairly concentrated sample to get a
reasonable absorbance (generally an absorbance between 0.1 and 1.0 is
considered within the “linear range”).
Also, ProtParam warns that there may be at least a 10% error in the
extinction coefficient if there are no tryptophans in your protein. Therefore, if the extinction coefficient is
low, which is likely the case if there are no tryptophans in the sequence, a
10% error could significantly throw off the assessment of the final protein
concentration.
Bradford Assay
How it works:
The Bradford assay
is a colorimetric assay based on the interaction between Coomassie brilliant
blue and the arginine and aromatic residues in the protein. When the dye binds to these residues, its
maximum absorption shifts from 470 nm to 595 nm. In general, we measure the absorbance of a
series of known concentrations of a standard protein, generally BSA, and create
a standard curve. We then use that
standard curve to calculate the concentration of your protein sample based on
its absorbance.
Advantages:
This assay is quick, and the reagent is not
affected by the presence of reducing agents.
Disadvantages:
Basic conditions
and detergents, such as SDS, can interfere with the dye’s ability to bind to
the protein; however, there are detergent-compatible Bradford reagents. Also,
like the absorbance at 280 nm technique, the Bradford assay depends on the
sequence of the protein. If the protein
doesn’t contain a decent number of arginine and/or aromatic residues, then the
dye will not bind to the protein as efficiently, resulting in an
underestimation of the protein concentration.
BCA Assay
How it works:
The BCA assay is
another colorimetric assay like the Bradford assay. It makes use of the biuret reaction, in which
the protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions. The Cu1+ ions then react with bicinchoninic
acid (BCA) to form a purple-colored product that absorbs at 562 nm. The procedure is similar to that of the
Bradford assay, in which we create a standard curve based on a series of known
protein standards.
Advantages:
Because the peptide
backbone is involved in the reaction, the BCA assay is less sensitive to the
types of amino acids in the protein.
However, the reaction is influenced by cysteine, tyrosine, and
tryptophan residues. The reagent is not sensitive to detergents and
denaturants, so it’s okay to have those in buffer.
Disadvantages:
The presence of
reducing agents in your buffer can interfere with the dye, but there are
reducing agent-compatible dyes available. The reaction takes some time to
proceed. Usually, the samples are
incubated at 37°C for 15-30 min. Also,
as in the Bradford assay, we determine the protein concentration by creating a
standard curve from a known, standard protein.
So again, if the protein doesn’t interact with the dye in a similar way
as the standard protein, the concentration could be off.
Spectrophotometer
calibration is a process in which a scientific instrument known as a
spectrophotometer is calibrated to confirm that it is working properly. This is
important, as it ensures that the measurements obtained with the instrument are
accurate. A spectrophotometer is capable of both transmitting and receiving
light. The device is used to analyze samples of test material by passing light
through the sample and reading the intensity of the wavelengths. Different
samples impact the light in different ways. Spectrophotometer calibration is
necessary to confirm that the results are accurate. In spectrophotometer
calibration, a reference solution is used to zero out the equipment. Blank
solution provides a base or zero reading. The device is calibrated by placing
the reference solution inside the spectrophotometer, zeroing out the settings,
and running the instrument. Then, samples of an actual test material can be
subjected to spectrophotometry in confidence that the machine has been
calibrated and is working properly.
References
