BIO 462
BIO 462
EXPERIMENT 2
EXPERIMENT 2
PROTEIN DETERMINATION
PROTEIN DETERMINATION
NAME: NURFADHILAH BINTI JAAFAR NAME: NURFADHILAH BINTI JAAFAR STUDENT ID: 2016675256 STUDENT ID: 2016675256 PROGRAM: PROGRAM: AS AS 246246 GROUP: 4B GROUP: 4B
DATE OF THE EXPERIMENT: 29
DATE OF THE EXPERIMENT: 29THTH MARCH 2017 MARCH 2017
DATE OF SUBMISSION: 5 APRIL 2017 DATE OF SUBMISSION: 5 APRIL 2017 LECTURER: MADAM AZANI BINTI SALEH LECTURER: MADAM AZANI BINTI SALEH
PROCEDURE
- Refer to Biochemistry Laboratory Manual page 5 until 14
-RESULTS 1. Biuret Method Blank: 0.612 Concentration of BSA OD (replicate 1) OD (replicate 2) OD (replicate 3) Mean 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 of BSA OD (replicate 1) OD (replicate 2) OD (replicate 3) Mean 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 of BSA OD (replicate 1) OD (replicate 2) OD (replicate 3) Mean 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
-Very high independent of composition of amino acid
- Good general protein assay for batches of material which yield is not a problem
-Involve single incubation for 30 minutes.
-Excellent
-More convenient than Lowry method
- Dependent on the amino acid composition of the measured protein. -Single incubation for 5 minutes - Fairly convenience - Can be performed at room temperature - Double incubation for 40 minutes. The first incubation is 10 minutes, second incubation is 30 minutes. Sensitivity -Least sensitive Bradford method and Lowry method
-0-1 mg small amount of protein can be detected
- More sensitive than Lowry method
-0-0.01 mg small amount of protein can be detected -Dependent on the composition of the amino acid - Sensitive to low protein concentration - Sensitive over a wide range - 0-0.1 mg small amount of protein can be detected Generality The consistency is quite fair
The consistency is good The consistency is excellent
Linearity
Linear Non- linear
concentration dependence
Non-linear concentration 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.
