LABORATORY 4: PROTEIN ANALYSIS

LABORATORY 4: PROTEIN ANALYSIS

OBJECTIVE

1.To determine protein concentration in various type of protein content

2.To determine protein concentration using two types protein assays that are Biuret assay and Lowry assay.

INTRODUCTION

There are many ways to measure protein concentration; one of the common protocol is by

measuring the absorbance of a coloured product formed by the protein and an organic

molecule. Different methods can yield somewhat different results for the same protein as

there is no absolute photometric protein concentration assay. All methods have advantages

and disadvantages and a few aspects will be taken into consideration: specificity, sensitivity,

the measurable range of concentration, the accuracy, the nature of the protein to be examined,

the presence of materials interfering with the measurement, and the time required for the

measurement.

Biuret protein assay use biuret reagent that detect the presence of peptide bonds.

When the peptide bonds present in an alkaline solution, the coordination complexes

associated with a copper ion (Cu2+) are violet in colour. Nitrogen atoms of the peptide bonds

form a coordination bond with metal ion. The quantity of the complexes formed is

proportional to the number of peptide bonds. Thus protein intensity affects the intensity of the

colour, where the colour will be more intense with more protein.

Figure 1. Biuret reagent is a blue liquid that changes to purple when proteins are present.

MATERIALS

Stock solution of Bovine Serum Albumin (BSA): 10mg/ml

Deionized water (dH2O)

Test tubes and stand

Pipette

Biuret reagent

Spectrophotometer

Protein samples

METHODOLOGY

(A) Preparation of biuret reagent

300ml of 10% (w/v) NaOH to 500 ml of a solution containing 0.3%  was added and stirred

copper sulfate pentahydrate and 1.2% sodium potassium tartarate, then 1 liter was diluted.

(B) Protein preparation

1. 2 sets of test tubes was prepared with the numbers 1 to 6 and prepare the bovine serum albumin

2. The duplicateof  test tubeswas prepared  for protein samples and carefully pipette 1ml of the protein

samples into each tubes.

Tube	BSA conc. (mg/ml)	H2O (ml)	BSA stock (ml)
1	0	1.0	0
2	1	0.9	0.1
3	2	0.8	0.1
4	3	0.7	0.3
5	4	0.6	0.4
6	5	0.5	0.5
7	6	0.4	0.6

3. 2ml of biuret reagent was added to every tube: the 14 tubes for the standard curve and the

duplicate tubes for protein samples.

4. The tubes was covered with parafilm and briefly vortex to ensure that the protein standards/

samples and the biuret reagent are thoroughly mixed.

5. The tubes was allowed  to stand at 15 minutes.

6. The spectrophotometer switched on and the wavelength adjusted to 550nm.

(C) Determine protein concentration

1.1 ml of solution from tube 1 was transferred into a cuvette and gently wipe the cuvette with a

paper towel to remove fingerprints and dust.

2. The absorbance was set up  to 'zero'. This tube will serve as 'blank'.

3. The absorbance of the other standards and sample proteins using the step as in (1) was measured.

*DO NOT blank the instrument again.

4. The absorbance of each standards and samples was recorded.

5. Graphof standard curve using the absorbance value of protein standards and

interpolate the absorbance values of the protein samples was plotted.

RESULTS

PROTEIN

PROTEIN I		PROTEIN II
SOY BEAN I	0.364	ALBUMIN I	0.579
SOY BEAN II	0.419	ALBUMIN II	0.513

SAMPLE OF PROTEIN

TUBE I		TUBE II
1	0.007	8	0.006
2	0.108	9	0.085
3	0.193	10	0.199
4	0.280	11	0.263
5	0.336	12	0.331
6	0.431	13	0.416
7	0.479	14	0.474

DISCUSSION

In a healthy urine sample, normal protein albumin concentrations are between 20ug/ml and 30ug/ml, this can be seen in Figure 2 with the Normal control having a value around 30 and for patient 3. However, protein levels from 30ug/ml to 300ug/ml are considered microalbuminuria; patient 1, 2, 4 and 5. The High Control Protein has a value in the 340 regions, this is known as macro albuminuria (Hasanato, 2016).Patient 1 – 35-year-old female. Albumin protein concentration is approximately 210ug/ml, abnormal level of protein is extremely higher than the normal range and can indicate preeclampsia. Women who experience preeclampsia during their pregnancy are more likely to experience complication than those with normal levels of protein, headaches are a clear indicator that the lady is showing signs of preeclampsia (Hawkins et al., 2015). It is essential that her blood pressure is monitored to prevent hypertension and sent for various blood tests to observe her kidney and liver function (NHS 2).Patient 2 – 50-year-old male. Albumin protein concentration is around 80ug/ml which is classed as microalbuminuria. Body mass index, waist circumference and high levels of protein in urine are linked with the formation of kidney stones (Garimella et al., 2015). The symptoms that patient 2 is experiencing suggest that he is suffering from a large kidney stone. Highly recommended that he is sent for blood and urine tests to check for infection (NHS 1).Patient 3 – 40-year-old female. Albumin Protein concentration is roughly 25ug/ml which indicates very little protein in her urine, concentration classed as normal as similar value to the Normal Control protein.Patient 4 – 23-year-old female. Albumin Protein concentration is in the region of 275ug/ml, classed as very high and could indicate albuminuria. Early diagnosis could prevent the patient from experiencing hypertension, type 2 diabetes and coronary heart disease (Yadav et al., 2016). The symptoms that the patient is experiencing could indicate a Urinary Tract Infection, there is a strong correlation in many studies that demonstrations how a UTI can impact the glomerular involvement increasing the levels of albumin in the urine (González Rodríguez et al., 2009).Patient 5 – 70-year-old. Albumin protein concentration boarding 60ug/ml which isn’t significantly higher the normal range but still classed as micro-albuminuria. Patient showing signs of still recovering from influenza, immune system is weakened due to age and can take longer to recover. Renal function decline can occur with age and within the elderly generation, which could indicate the abnormal levels of albumin in his urine (Funk et al., 2016). Should be monitored regularly, a review of his medication that may cause a decline in his renal function and put on antibiotics if symptoms of flu continue.

CONCLUSION

Through  this protein analysis experiment, we can determine protein concentration in various type of protein content and determine protein concentration using two types protein assays that are Biuret assay and Lowry assay.

REFERENCES

·        Top 10 Protein Supplements. (n.d.). Retrieved from https://labdoor.com/rankings/protein

·       Dlyakh783. (n.d.). Protein Lab Report !! - Calculating the Amount of Protein... Retrieved from https://www.coursehero.com/file/18935224/Protein-Lab-Report-/