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Experiment 2: Protein Experiment
Introduction:
There is no single protein assay method
that yields absolutely accurate results. The only true and accurate method for
determining protein concentration is by acid hydrolyzing a portion of the
sample and then carries out amino acid analysis on the hydrolyzate. But, this
method is time-consuming. Each method and assay has its own disadvantage and limitations. In this experiment, Biuret assay
and Lowry assay method will be use to determine protein concentration. The
spectrophotometer is used to measure the amount of light that a sample absorbs.
Objectives:
1.
To determine protein concentration of the unknown
protein solution
2. To draw standard curve by plotting the absorbance
(nm) against the concentration of protein standard solution (mg/mL).
Materials:
1.
Prepared solutions of gelatin at 1, 2, 3, 4, 5, and
6 mg/mL in water à Biuret assay
2.
Prepared 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL à Lowry assays
3.
Biuret reagent
4.
Lowry reagent 1
5.
Lowry reagent 2
6.
Protein
Procedures:
Burette
Assays:
1) Firstly
the gelatine solutions were prepared at the concentrations of 1, 2, 3, 4, 5 and
6 mg/ml.
2) Then,
we take out only 0.5ml of the protein from 1 mg/ml volumetric flask.
3) Then
it was being mixed with 2.5ml of the Burette reagent.
4) After
that it was left for 10 minutes and then being measured the absorbance.
5) Other
concentrations are being repeated with the same steps.
Lowry
Assays:
1) The
concentration of the protein was prepared under 50 times dilution which is
0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 mg/ml.
2) Next,
only 0.25ml of the protein being taking out and put into the test tube.
3) Then,
it being mix with another 2.5ml Lowry Reagent 1.
4) After
10 minutes, the mixture being added with the Lowry Reagent 2 and being left for
30 minutes.
5) After
the time allocated finish so we could measured the absorbance value.
Results
Sample
Type of Eggs
|
Absorbance Value(nm)
|
Concentrations(mg/ml)
|
Real Value (50x dilutions)(mg/ml)
|
Telur biasa
|
0.199
|
2.5
|
125
|
Kampong
|
0.190
|
2.3
|
115
|
Puyuh
|
0.175
|
2.1
|
105
|
Itik
|
0.183
|
1.9
|
95
|
omega
|
0.162
|
1.5
|
75
|
STANDARD
SOLUTION
BIURET ASSAY
Concentration
(mg/mL)
|
Absorbance
(nm)
|
1
|
0.154
|
2
|
0.180
|
3
|
0.218
|
4
|
0.229
|
5
|
0.289
|
6
|
0.426
|
LOWRY ASSAY
Concentration
(mg/mL)
|
Absorbance
(nm)
|
0.1
|
0.070
|
0.2
|
0.125
|
0.3
|
0.065
|
0.4
|
0.130
|
0.5
|
0.108
|
0.6
|
0.155
|
Discussion
Biuret assay and lowry assay is a method used to
determine the concentration of protein. Absorbance at 540 nm for the Biuret and
750 nm for the Lowry assays. Both need alkali condition for method to be
appropriate. Biuret test detect the presence of peptide bond. Peptide bond occurs with the same frequency per amino acid in the
peptide. The intensity of colour is directly proportional with the protein
concentration. The spectrophotometer was used to. Measure the intensity
of the color produced. When the protein concentration is higher it will turn
the colour to darker. The colour changes because biuret reagent was react with
the peptide bond. Lowry assay lies in the
reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline
conditions and the subsequent reduction of the Folin-Ciocalteay
phosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by the
copper-catalyzed oxidation of aromatic acids. The Lowry method is sensitive to
pH changes so for this method to be appropriate it needs alkali condition.
Other than that lowry method is sensitive to low concentrations of protein.
The other method to determine the protein
concentration by ultraviolet (UV) absorbance at 280 nm for range 0.1-100 ug/ml and Bradford or Coomassie
brilliant blue protein assay for range 20-2000 ug/ml . ultraviolet (UV)
absorbance at 280 nm ,Aromatic amino acids tyrosine and tryptophan give
proteins the characteristic ultraviolet (UV) absorption spectrum at 280 nm.
Phenylalanine and disulfide bonds also contribute the absorption at that
wavelength, albeit slightly. This method is quickest but error prone and
requires an extremely small sample volume since new spectrophotometers employ a
sample retention system during the measurement.The protein sample should be
pure and does not contain any non-protein components with the same absorption
spectrum, such as the contamination of nucleic acids.
Bradford or Coomassie brilliant blue protein assay
,it relies on the formation of a complex between Coomassie brilliant blue G-250
dye and proteins in solution. The free dye exists in four different ionic
forms. The more anionic blue form binds to proteins and has an absorbance at
590 nm. The protein concentration can be evaluated by determining the amount of
dye in the blue ionic form and by measuring the absorbance of the solution at
595 nm using a spectrophotometer . The dye binds mostly to arginine,
tryptophan, tyrosine, histidine, and phenylalanine residues of the protein. One
advantage of this method is its compatibility with reducing agents used to
stabilize proteins in solution, which are not compatible with Lowry assay and
in some extent with the BCA assay. The limitation of Bradford assay is the
incompatibility with low concentration of detergents, which are routinely used
to solubilize membrane proteins. , one disadvantage of using BSA is that it
exhibits a strong dye response and may under-measure the protein content.
Appropriate blank is used for zeroing the spectrometry. It is important to
measure absorbance that due to the present of solute of interest
Conclusion
In
this experiment, we wished to discover the identities of different types of egg
dilution by analyzing their protein concentrations through spectrophotometry. Since
the dye we used changed to darker colors when in contact with a higher protein
concentration, a higher absorbance meant a higher concentration. From this
experiment, we were able to estimate the protein concentrations of our different
of types of egg dilution and compare to the standard solution that already have
been diluted. For the Biuret assay telur biasa have the highest real value
which is 125mg/ml, followed by telur kampung 115mg/ml, telur puyuh 105mg/ml,
telur itik 95mg/ml and omega 75mg/ml. However, in Lowry assay we does not
obtain the result.
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