DETERMINATION OF PROTEIN CONCENTRATION FROM LIQUID MILK USING

BRADFORD ASSAY

ABSTRACT (5)
The Bradford assay place reliance on the binding of Coomassie Brilliant Blue G250 dye to
protein to determine their concentration. The binding of the reagent to proteins
causes a switch from acidic conditions where the dye is primarily in the doubly
protonated red cationic form at an absorbance of 470nm to a stable unprotonated
blue
form at an absorbance of 595nm. The detection of blue protein-dye form at 595nm is
observed through spectrometer in an assay. The Bradford assay bring immediate
absorbance which makes the process fast with a rapid color development and no
meddling of reagents besides detergents. The increase in proteins in the assay
increases
the density of the color which becomes bluer. The curve may not be a linear due to
occurrence of errors.

INTRODUCTION (10)
Proteins are building blocks of all life forms. Plants and animals contain proteins in a
form of enzymes, tissues, hairs and the list go on. Protein concentration can be
conveniently measured by Bradford assay. The Bradford assay is quick, less costly and
it is identified as the easy way to determine the concentration of protein compared to
to other methods. The methods are Lowry method, Biuret method, Bicinchoninic acid
assay and colloidal gold protein assay. The Bradford assay is designed to find total
concentration of proteins in a solution. It is best used for determining protein
concentration for cell fractions and gel electrophoresis, but more frequently it is used
for protein concentration. The Bradford assay uses a reagent called Coomassie
Brilliant
Blue G-250. The reagent is reacted with proteins and use the spectrometer to
measure
the absorbance. The CBBG dye contains two sulfonic acid groups and six phenyl
groups,
which interact with proteins via positively charges residues by using Van der Waals
forces the and hydrophobic residues, respectively. There are also ionic interactions.
The
CBBG and protein reaction is preserved bye these forces. The reaction take place in an
acidic medium. The absorbance increases from 470nm to 595nm once it forms a
complex with a protein. The absorbance differs with difference in concentration and
forms a linear regression equation which can be obtained by drawing a graph of
absorbance versus amount of protein. The assay forms a color change as the
as the amount of protein increases, this color change is due to the binging of proteins
and the dye. The spectrometer is used since it can produce light of a curtain
wavelength
And monitor the light intensity.

MATERIALS & METHODS (10)

*Spectrophotometer measuring absorbance in the 595 nm region.
*1X PBS
*3 mL Disposable Plastic Cuvettes
*1X Bradford reagent
*Protein Standard (BSA) Solution, (2 mg/mL)
*20-200 microliter/micropipette
*Milk
*Pipette tip
*Microcentrifuge tube
*2-20 microliter/micropipette
*Cuvette tips
*Microcentrifuge
 Using a 20-200 microliter/micropipette; the pipette was set to 98 microliter and
pipetted 1x PBS into an empty microcentrifuge tube labelled sample A. Switched the
pipette to a 2-20 microliter/micropipette and set the pipette to 2 microliters. Pipetted
2 microliters of milk sample into a microcentrifuge labelled sample A. Labelled 2
cuvettes 1 control and the other sample. Pipetted 20 microliters of diluted milk
sample
into the cuvette labelled sample and 20 microliters of 1X PBS into a cuvette labelled
control. The protein standards were set ranging from 0.125mg/ml till 2mg/ml used a
BSA
as standard solution. Cuvettes were labelled for each standard and marked with
corresponding concentration. 20 microliters of each protein were added
corresponding
to the cuvette. For each sample a new clean pipette tip was used. Added Bradford
reagent to all the cuvettes that had been prepared including control and sample.
Mixed
well by vortexing. The cuvettes were incubated at room temperature for 5 minutes
were visually compared the milk sample to the protein standard to determine which
one closely matches the color milk sample. The protein concentration was visually
was visually estimated. Using a spectrophotometer, Bradford assay was selected and
set blank. Control sample was inserted to the spectrophotometer and used to set it to
absorbance using an A595. Read absorbance to the 7protein standards and recorded
the values. Lastly removed the cuvette labelled blank and inserted milk sample into
the
spectrophotometer and read the absorbance. The values were recorded.

RESULTS (10)

Table 1: Absorbance values observed and the working concentration for BSA

Absorbance values observed

Control (1X PBS + Reagent) 0.558nm
Milk + Reagent 1.472nm
BSA + Reagent (0.125 mg/ml) 0.625nm
BSA + Reagent (0.25 mg/ml) 0.666nm
BSA + Reagent (0.5 mg/ml) 0.827nm
BSA + Reagent (0.75 mg/ml) 0.888nm
BSA + Reagent (1 mg/ml) 0.935nm
BSA + Reagent (1.5 mg/ml) 1.165nm
BSA + Reagent (2 mg/ml) 1.226nm

The absorbance of sample B that has milk and reagent obtained was higher than all
the
Absorbance obtained.

Figure 1: The graph representing a protein standard curve.

 Absorbance value vs Bradford standard concentration
1.4

f(x) = 0.330112359550562 x + 0.615723113964687

1.2 R² = 0.966980883990289
Absorbance value observed (nm)A595

0.8
Absorbance value observed
Linear (Absorbance value observed)
0.6

0.4

0.2

0
0 0.5 1 1.5 2 2.5

Cuvettes concentration(mg/ml)

DISCUSSION (8)
For sample A
0.558 = 0.6157x + 0.3301
0.6157x = 0.2279
X = 0.370 mg/ml approximate concentration
Sample B
1.472 = 0.6157x + 0.3301
0.6157x = 1.1419
X = 1.8546 mg/ml approximate concentration
With 7 standards there was an increase in the density of the color change from red
to pure blue. The color of the standards was directly proportional to their
concentration hence the absorbance increased as the concentration increased as
shown in table 1 above. The increase in concentration brought the color change from
red to blue. Sample A and sample B has an absorbance close to standard 3 and
standard
7 respectively. The concentration of sample A and sample B were also calculated and
it
was discovered that they also correspond with concentration of standard 3 and 7
Respectively. Sample A has a concentration of 0.370mg/ml and sample B has a
Concentration of 1.8546mg/ml. There results were obtained in consideration of error
that might have occurred.

CONCLUSION (2)
The results showed that Samples A and B had concentrations near that of Standard 3
and standard 7. The results also showed that the increase in concentration of protein
increased the absorbance and the color changed from red to blue.

REFERENCES (4)
1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical
Biochemistry, 72(1), 248-254.
2. Stoscheck, C. M. (1990). Quantitation of protein. Methods in enzymology, 182, 50.
3. Bradford Protein Concentration Assay.(2001). Retrieved September 23, 2008 from
http://www.acad.carleton.edu/curricular/BIOL/resources/rlink/lab2p2.html.
4. Caprette, David R. (May2005a). Bradford protein assay, from Experimental
Biosciences Website:http: //web.chemistry.gatech.edu/~williams/bCourse_
Information/4581/techniques/bradford/bradford.htm