Protocol on Proteins determination using a modified Folin-Lowry assay PDF

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Protocol on Proteins determination using a modified Folin-Lowry assay (Up839733 and UP816084)

Figure 1. This diagram shows the redox reaction of copper and peptide bonds from proteins1 Lowry protein assay is a method used to determine protein concentration, the mechanism of the Folin-Lowry assay is based upon 2 reactions, the Biuret reaction and the Lowry reaction. Biuret reaction involves protein peptide bonds to react with the Cu2+ in alkaline conditions. This causes the reduction of Cu2+ ion to Cu+ and the oxidation of the peptide bonds, additionally the Cu+ ion undergoes a further reaction called the Lowey reaction. Figure 1 demonstrates how proteins react with copper ions. Lowry reaction occurs when the Cu+ ion react with the Folin-Ciocalteu reagent, where phosphomolybdotungstate is reduced to heteropolymolybdenum blue. Furthermore, this forms a blue purple colour which is dependent on the concentration of aromatic amino acids present2.

Reagents used: 

50mgmL-1cupric sulphate stock solution-50ml



0.9% sodium chloride (saline)-100ml



1% (w/v) aqueous sodium potassium tartrate-100ml



5mg/ml Bovine serum albumin (BSA)



10% (w/v) anhydrous sodium carbonate in 0.5M sodium hydroxide-50ml



Folin-Ciocalteu reagent, 2 %( v/V) in distilled water-250ml

Equipment required: 

Glass beakers



Measuring cylinders



Gloves



Lab coat



Safety goggles



Spectrophotometry



Glass test tubes



Test tube rack



P200, P1000, P5000 Gilson pipettes and tips



Biohazard bag



Gloves



Tissue

1



Tube racket



50ml and 100ml Volumetric flask



Funnel



Glass beaker



Vortex Mixer



Stop watch

Procedure: In the following laboratory experiment, you will be working in pairs to produce a protein assay using the modified Folin-Lowry method. You then will use this protein assay to estimate the albumin concentration of 3 unknown patient samples. Part 1: Preparation of cupric sulphate diluent 1. Measure out 75ml of 1% (w/v) aqueous sodium potassium tartrate using a measuring cylinder and then pour into a 100ml volumetric flask 2. Pour 25ml of 10% (w/v) anhydrous sodium carbonate in sodium hydroxide 0.5M into the flask ensuring the line is reached 3. Stir the volumetric flask for 2 minutes 4. Carefully use a funnel to pour this cupric sulphate dilute into a glass beaker 5. Label this beaker cupric Sulphate diluent

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Part: 2 Preparation of cupric sulphate solution 1. Pipette 1ml of 50mg/ml cupric sulphate stock solution into a 100ml volumetric flask 2. Measure out 99ml of cupric sulphate diluent using a measuring cylinder then add to the volumetric flask making sure that the solution reaches the line 3. Stir and invert the volumetric flask for 2 minutes 4. Carefully use a filter to pour this cupric sulphate into a glass beaker 5. Label this beaker Sulphate solution Part 3: Protein Assay Procedure Table 1: Volume of BSA and deionised water required to generate BSA standards with a finial volume of 1.0mL Standard

Volume of BSA

Volume of DI h20

Concentration of

stock solution (ml)

(ml)

BSA (mg/ml) 0 (Blank) 0.5 1.0 2.0 3.0 4.0

0.1 0.2 0.4 0.6 0.8

1.0 0.9 0.8 0.6 0.4 0.2

Total volume (ml)

1.0 1.0 1.0 1.0 1.0 1.0

1. Collect 6 test tubes and label each one with each standard concentration listed on table 1. 2. Following table 1, pipette each of the different BSA stock solution and deionisation water into each test tube respectively.

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3. Place each test tube on the vortex mixer for 2 minutes 4. Collect 6 more test tubes 5. Pipette 0.1ml of the standard solution into a new test tube and then pipette 0.9ml of 0.9% (w/v) saline. 6. Label the test tube the corresponding standard concentration used 7. Repeat step 1 and 2, for all the other standard concentration and blank 8. Pipette 0.05 ml of a patient serum sample into a new test tube 9. Pipette 0.95ml of 0.9% (w/v) saline 10. Pipette out 0.1ml of this solution into another test tube 11. Pipette 0.9ml of 0.9% (w/v) saline and label the patient number 12. Repeat until all the 3 sample patient solutions are made 13. Pipette 1ml of cupric sulphate solution to all the patient samples and standard concentrations 14. Then incubated all the test tube for 10 minutes at room temperature (25oC) 15. Following the incubation period, add 3ml of Folin-Ciocalteu reagent (2 %( v/v) in distilled water to all the test tubes 16. Then incubate at room temperature for a further 30 minutes. 17. Switch the absorbance on the spectrophotometer to 660nm. 18. Pipette 1ml of the blank into a cuvette and set to zero.

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19. Starting with the lowest concentration of BSA, pipette 1ml of the solution into a cuvette and measure the absorbance using the spectrophotometer. 20. Generate a mean standard concentration for BSA, and construct a standard curve 21. Measure the absorbance for the 3 patient samples and estimate the concentration of albumin using the calibration curve. Results Table 2. BSA standard concentration repeats from 0.5 to 4.0 mg/ml Concentration

Absorbance

Absorbance

Absorbance

Absorbance

Mean

of albumin

of 1st series

of 2nd series

of 3rd series

of 4th series

absorbance

(mg/ml) 0.5 1.0 2.0 4.0

0.132 0.300 0.484 0.736

0.146 0.264 0.503 0.742

0.105 0.211 0.390 0.620

0.104 0.215 0.385 0.616

0.122 0.248 0.441 0.679

Absorbance

Table 3. Patient sample repeats Patient

Absorbance

Absorbance

Absorbance

samples 1 2 3

of 4th series 0.473 0.266 0.401

of 2nd series 0.398 0.285 0.387

of 3rd series of 4th series absorbance 0.457 0.406 0.434 0.278 0.297 0.282 0.405 0.409 0.401

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Mean

Calibration curve of concentration of albumin against absorbance 0.8 0.7

f(x) = 0.17 x + 0.05 R² = 0.97

Abs 660nm

0.6 0.5 0.4 0.3 0.2 0.1 0

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

BSA concentration (mg/ml)

Figure 2. Standard curve BSA concentration against Abs 660nm

Table 4. Patient sample estimated albumin concentration in g/dL and then to adjust from the 1:20 ratio dilution, the concentration is times by 20 Patient number

Concentration of albumin (g/dL) 4.62 8.08

1 2 3

4.24

Discussion

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Albumins levels give indication of the body not functioning properly as high or low levels of albumin are usually a symptom of other diseases (6). The reference range of albumin for healthy humans is between 3.5 to 5.5 g/dL (4). From Table 4 we can see that the concentration of albumin in patient 1 and 3 is between the normal ranges but the concentration of albumin in patient 2 is higher. High levels of albumin in medical terms is called hyperalbuminemia. Hyperalbuminemia can be caused by many condition such as dehydration, acute infections, cancer, chronic inflammatory disease (5) and liver diseases such as hepatitis or cirrhosis (4). Hepatitis can arise from viral infection or liver damage due to drinking alcohol. Alcoholic hepatitis can lead to the development of Cirrhosis which can lead to liver failure or cancer.

Validation The Lowry assay is sensitive to low concentration of protein, one of the earlier study by Dunn [1992] suggested using concentration between the ranges of 0.005-0.1mg/ml. Although, a more recent study suggested 0.10-2mg. To reduce a sensitivity error, a narrower range of standard concentration is recommended and more standard concentration from the lower range of 0.05-2mg /ml should be used. As you can see from table 5, the results were more concentrated and darker than our highest concentration which was 0.679. They exceed the ranges for our standard calibration curves. If we extrapolate these data, we will end up with a less accurate estimation. For this reason, we voided the set of results from table 5 and adjusted the experiment by diluting the patient sample by 1:20. Therefore, we diluted the patient sample by 1 to 20 ensuring that the patient sample absorbance is within the acceptable range. 7

Figure 2 has an R2 value of 0.9711 which is very close to 1. This shows that the date is very close to the fitted regression line increasing the validity of the results. To make the results more reliable, duplicates of the solution were made, and their absorbance was measured. Then an average of their absorbance was taken which reduces the chance of random errors. Considering the experiment requires incubation at room temperature, our experiment occurred without a water bath. However, due to changing room temperatures throughout the day, this may not be a suitable outcome. To make this experiment more reproduceable, a water bath is suggested.

Health and safety were taken into considerations as all the safety measures were taken to carry out this experiment in the safest way possible. Gloves, goggles and laboratory coats were worn throughout the experiment to prevent the solutions getting onto the skin and eyes as some of the solutions were corrosive and toxic. [Word count 1077]

Appendix Each person from a pair will do a duplicate of each standard concentration, this will produce 4 results for the standard concentrations and patient samples. Cupric sulphate solution: 1 blank, 5 standards and 3 test sample are required to make 4 repeats,

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4(5x2x1 ml) +1+4(3x2x1ml) =65ml, therefore 100ml should be made. To calculate how much reagent is required to make 100ml of cupric sulphate solution, dilution factor equation (C1V1=C2V2) is used. This combines two appropriate volumes to produce the required concentration3. C1=Concentration of Cupric sulphate stock solution (50mgmL-1) V1=Volume of Cupric sulphate stock solution (x) C2= Concentration of finial cupric sulphate solution (0.5mgmL- 1) V2= Volume of finial cupric sulphate solution (100mL) C1V1=C2V2, 50x=0.5(100), V1 = 1ml To get the dilution volume you use V2 - V1= 100ml-1ml =99ml Therefore, to make the finial cupric sulphate solution, 1ml cupric sulphate stock solution and 99ml of the cupric sulphate diluent is required. Preparation of standard protein solutions Example 1. Find how much BSA solution and deionisation water is needed to make a 1ml BSA standard concentration for 2 mg/mL C1= Concentration of stock solution (5mg/ml) V1= Volume of BSA stock solution (x) C2= Finial concentration of standard (2 mg/mL) V2= Finial volume of standard solution (1mL) C1V1=C2V2, 5x=2(1), V1=0.4ml 9

Therefore, 0.4ml of 5mg/ml-1 BSA and 0.6ml deionised is required to make 1ml of 2 mg/mL

Estimation of a patient samples albumin concentration Patient 1 has a mean absorbance of 0.434. Using the calibration curve of concentration of albumin against absorbance graph, we know that the equation is y = 0.1672x + 0.0472. Y=0.434 0.434=0.1672X+0.0472 X= (0.434-0.0472)/0.1672 X=2.313 Table 5. Patient sample results without the 1:20 dilution with saline Patient

Absorbance

Absorbance

Average

samples 1 2 3

of 1st series 1.087 1.036 1.075

of 2nd series 1.047 1.050 1.111

1.067 1.043 1.093

These results looked more concentrated and darker than our highest concentration which was 0.679. They exceed the ranges for our standard calibration curves. If we extrapolate these data, we will end up with a less accurate estimation. Therefore, we diluted the patient sample by 1 to 20 ensuring that the patient sample absorbance is within the standard curve graph. Table 6. Conversion of units and adjustments

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Patient number

Concentration of

Concentration of

Concentration of

albumin in mg/ml

albumin in g/dl

albumin adjusted from 1:20 ratio

1

2.313

0.231

4.62

2 3

1.404 2.116

0.404 0.212

8.08 4.24

References 1.

Chemistry of Protein Assays | Thermo Fisher Scientific [Internet]. Thermofisher.com. 2017 [cited 3 November 2017]. Available from: https://www.thermofisher.com/ar/en/home/life-science/protein-biology/proteinbiology-learning-center/protein-biology-resource-library/pierce-proteinmethods/chemistry-protein-assays.html

2.

Construction of Protein Standard Curve using Folin’s Lowry Method (Theory) : Biochemistry Virtual Lab II : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab [Internet]. Vlab.amrita.edu. 2017 [cited 2November 2017]. Available from: http://vlab.amrita.edu/? sub=3&brch=64&sim=1087&cnt=1

3.

Making Simple Solutions and Dilutions [Internet]. Abacus.bates.edu. 2017 [cited 31 October 2017]. Available from: http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html

4. Devaraj, S. (2017). Albumin: Reference Range, Interpretation, Collection and Panels. [online] Emedicine.medscape.com. Available at:

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https://emedicine.medscape.com/article/2054430-overview#a2 [Accessed 16 Nov. 2017]. 5. Tandurust.com. (2017). High Albumin Levels: Causes, Symptoms Of Elevated Albumin In Blood. [online] Available at: http://www.tandurust.com/health-faq-7/albumin-high-levelscauses.html [Accessed 16 Nov. 2017]. 6. Labtestsonline.org.uk. (2017). Albumin: The Test. [online] Available at: http://labtestsonline.org.uk/understanding/analytes/albumin/tab/test/ [Accessed 16 Nov. 2017].

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