Title:Spectrophotometry and protein concentration measurement

Material:
-Stock solution of Bovive Serum Albumin (BSA): 10mg/ml
-Deionized water (dH2O)
-Test tubes and stand
-Pipette
-Biuret reagent
-Spectrophotometer
-Protein samples(fish and soy bean)

Procedure

A) Preparation of biuret reagent

Added, with stirring, 300ml of 10% (w/v) NaOH to 500 ml of a solution
containing 0.3% copper sulfate pentahydrate and 1.2% sodium potassium
tartarate, then diluted to 1 liter

(B) Protein preparation

1. Prepared 2 sets of test tubes with the numbers 1 to 6 and prepared the
bovine serum albumin (BSA) stock solution (10 mg/ml) according to the
concentration listed below:

Tube BSA conc. H2O BSA stock total Biuret

(mg/ml) (ml) (ml) reagent
1 0 1.0 0 1 2
2 1 0.9 0.1 1 2
3 2 0.8 0.2 1 2
4 3 0.7 0.3 1 2
5 4 0.6 0.4 1 2
6 5 0.5 0.5 1 2
7 6 0.4 0.6 1 2
2. Prepared duplicated test tubes for protein samples and carefully pipette 1ml
of the protein samples into each tubes.
3. Added 2ml of biuret reagent to every tube: the 14 tubes for the standard
curve and the duplicate tubes for protein samples.
4. Cover the tubes with parafilm and briefly vortex to ensure that the protein
standards/ samples and the biuret reagent are thoroughly mixed.
5. let the tubes to stand at 15 minutes.
6. Switch on the spectrophotometer and adjusted the wavelength to 550nm.

(C) Determine protein concentration

1. Transfer 1ml of solution from tube 1 into a cuvette and gently wipe the
cuvette with a paper towel to removed fingerprints and dust.
2. Set the absorbance to 'zero'. This tube will serve as 'blank'.
3. Measured the absorbance of the other standards and sample proteins
using the step as in (1). *DO NOT blank the instrument again.
4. Recorded the absorbance of each standards and samples.
5. Plot a graph of standard curve using the absorbance value of protein
standards and interpolate the absorbance values of the protein samples.

Result
 No Absorbance Concentration
1 0 0
2 0.082 1
3 0.175 2
4 0.210 3
5 0.270 4
6 0.352 5
7 0.522 6
Sample A(soy bean) 0.676
Sample B(fish) 0.751

Graph absorbance againts concentration of sample

Discussion

While performing experiments there are some errors that directly affects the
experiment changed.maybe this is because of the test tube was not clean
before start the experiment and the data it will give wrong value if there was
fingerprint at the tube.We need to clean the test tube and tube properly,so it
will make no error in our experiment.
Next,during the experiment we need to use Spectrophotometer to see
absorbance for each sample,but before put the tube inside the
Spectrophotometer we need to clean it properly such as fingerprint or etc.
Other than that,the graph shown the value of standardise sample and sample
A and B,form the graph absorbance againts concentration we can get the
concentration of sample which is make a straight line like a graph above.

Conclusion

The conclusion is fish contain more protein than soy bean.we better eat fish
more than eat soy bean.

Reference
1.https://www.ncbi.nlm.nih.gov/pubmed/18228395

2.http://onlinelibrary.wiley.com/doi/10.1002/0471142913.fab0103s04/abstract