BIO 462

EXPERIMENT 2
PROTEIN DETERMINATION

NAME: NURFADHILAH BINTI JAAFAR

STUDENT ID: 2016675256

PROGRAM: AS 246

GROUP: 4B

DATE OF THE EXPERIMENT: 29TH MARCH 2017

DATE OF SUBMISSION: 5 APRIL 2017

LECTURER: MADAM AZANI BINTI SALEH

PROCEDURE

- Refer to Biochemistry Laboratory Manual page 5 until 14 -

RESULTS
1. Biuret Method
Blank: 0.612
Concentration OD OD OD Mean
of BSA (replicate 1) (replicate 2) (replicate 3)
2mg/ mL 0.345 0.338 0.339 0.3406
4 mg/ mL 0.411 0.413 0.413 0.412
6 mg/ mL 0.406 0.408 0.405 0.406
8 mg/mL 0.400 0.398 0.401 0.400

2. Bradford Method
Blank: 0.727
Concentration OD OD OD Mean
of BSA (replicate 1) (replicate 2) (replicate 3)
0.1 mg/ mL 0.818 0.817 0.816 0.817
0.2 mg/ mL 0.848 0.847 0.847 0.847
0.3 mg/ mL 0.915 0.916 0.917 0.916
0.4 mg/mL 1.045 1.045 1.042 1.044

3. Lowry Method
Blank: 0.082
Concentration OD OD OD Mean
of BSA (replicate 1) (replicate 2) (replicate 3)
0.1 mg/ mL 0.253 0.250 0.247 0.250
0.2 mg/ mL 0.288 0.290 0.288 0.289
0.3 mg/ mL 0.348 0.346 0.348 0.347
0.4 mg/mL 0.304 0.300 0.303 0.302
EVALUATION OF EACH METHOD BY THE CRITERIA

1. Convenience (how easy is it to do?)

2. Sensitivity (how well does it detect small amounts of protein?)
3. Generality (how consistent are the results among different proteins?)
4. Linearity (does it give straight line plot of absorbance vs protein?)

Biuret Bradford Lowry

Convenience
- Good -Excellent - Fairly
-Very high independent convenience
of composition of -More convenient than - Can be performed
amino acid Lowry method at room
temperature
- Good general protein - Dependent on the - Double incubation
assay for batches of amino acid composition for 40 minutes.
material which yield is of the measured protein. The first
not a problem incubation is 10
-Single incubation for 5 minutes, second
-Involve single minutes incubation is 30
incubation for 30 minutes.
minutes.

Sensitivity
-Least sensitive - More sensitive than - Sensitive to low
Bradford method and Lowry method protein
Lowry method concentration
-0-0.01 mg small - Sensitive over a
-0-1 mg small amount amount of protein can wide range
of protein can be be detected - 0-0.1 mg small
detected amount of protein
-Dependent on the can be detected
composition of the
amino acid
Generality

The consistency is The consistency is good The consistency is

quite fair excellent

Linearity

Linear Non- linear Non-linear concentration

concentration dependence
dependence
DISCUSSIONS

The determinations of protein concentration are based on the amount, nature of protein to
be analyzed, the presence of interfering substances, the sensitivity of the equipment and the
method used. In this experiment, the protein in BSA concentration was determined by using the 3
methods which are Biuret Method, Bradford method and Lowry method and employ with
spectrophotometer. The graph absorbance against concentration for the 3 types of method
constructed as shown on the result section from the samples prepared.

The determination of protein measured by the amount of light that the sample absorbs. The
first method that was carried out is Biuret method and tested with the 2,4,6,8 mg/ mL concentration
of BSA and the spectrophotometer average reading shows 0.3406, 0.412, 0.406 and 0.400
respectively. The value of the absorbance slightly fluctuate and decreasing. The blank solution that
contain only Biuret reagent shows 0.162 on the spectrophotometer. The blank is put as it is act as a
reference. Cu2+ ion that present in the Biuret reagent form a complex with peptide bonds of the
protein and it should be resulted with the most linear result. However, the colour of the complex
might be somewhat different depending on the concentration of the protein.

The result indicate that the BSA concentration for 0.1, 0.2, 0.3 and 0.4 mg/mL when used
the Lowry method comes with values of the average absorbance of 0.250, 0.289, 0.347 and 0.302
respectively and the value of the blank is 0.082. The value of the absorbance decrease slightly at
BSA concentration of 0.4 mg/mL. The result was observed that Lowry method is more sensitive
than Biuret method. It is based on the reaction of Cu+ produce by the oxidation of peptide bonds
with the Folin-Ciocalteu reagent.

The Bradford method used the BSA concentration with lower amount of concentration with
0.1, 0.2, 0.3, and 0.4 mg/mL and resulted with the average absorbance value of 0.817, 0.847, 0.916
and 1.044 respectively. The blank sample is 0.727. It shows that the lower the amount of
concentration gives the lower reading of the absorbance. The method is dependent on the amino
acid composition of the measured protein and it is more efficient, than other method as it involves
fewer mixing steps, no heating required and it gives more stable colorimetric response. It is proved
by comparing the result from the 3 methods mentioned before; graph by the Bradford method is
directly proportional with the absorbance against the concentration. There is no fluctuation value of
the absorbance when using the Bradford method compared with the Lowry and Biuret method.
 A lot of factors can cause the error in the experiment and classified based on the 2 types of
error which are random error and systematic error. The spectrometer may not be calibrated
properly or it happened when the pipetting the reagents and the dye which can cause problem such
as inaccurate mixing and addition of the solutions. Spectrophotometer should be down to the zero
point by the reagent blank since it can be a very big factor for error in the experiment. Besides that,
the random error can occur due to the personal error for instance when the test tube used are not
clean enough or due to the inaccurate amount of volume of solution which cause of inadequacy
and slightly diluted of the solution prepared.

CONCLUSIONS

In conclusion, the Bradford technique appears to be the best method in determining the
protein determination. The objectives of the experiment to determine the protein by using Biuret,
Lowry and Bradford method by using the spectrophotometer was achieved. Bradford method is the
most convenient compared to Biuret and Lowry method as Bradford method gives accurate
reading, simple, fast and high sensitivity. It also shows that the graph of the absorbance is directly
proportional against the BSA concentration without any fluctuation value.