enzymology

Experiment 2

Aim of the Experiment

Quantitative estimation of proteins by Bradford/Lowry’s method.

Principle

1. Folin – Ciocalteau (Lowry) Assay for Protein Estimation:

Principle: The Lowry’s method utilizes phenol reagent of Folin and Ciocalteau. This is essentially phosphotungstic phosphomolybdic acid which can be reduced by phenols and many other substances with phenolic rings to ‘molybdenum blue’. Proteins reduce phenol reagent, which may be used therefore for their determination. However, the amount of colour varies greatly with different proteins because it is entirely proportional to their content of tyrosine and tryptophan, other amino acids having little effect. Pretreatment of proteins with alkali and a trace of copper salt greatly increases the colour that is absorbed maximally at 750 nm.

2.Bradford Assay for Protein Estimation:

Principle: Bradford Assay for protein estimation is a rapid, simple and sensitive method for estimation. The dye used in this method, Coomassie brilliant Blue G – 250 has an absorbance maximum of 465 nm in unbound state. When it interacts with proteins, the dye turns blue in colour and the complex formed has a max of 595 nm. Thus, proteins can be estimated at 595 nm and the colored complex formed is stable for one hour.

Materials required

Chemical

1. Prepared crude extract,

2. 40% pellet,

3. dialyzed sample,

4. BSA standard solution (1mg/ml),

5. Folin-Ciocalteu reagent,

6. Reagent A,

7. Reagent B,

8. Reagent C,

9. Distil water.

Equipment and Glassware

1. Test tubes,

2. test tube rack,

3. pipette,

4. pipette pump,

5. plastic cuvettes,

6. spectrophotometer.

Procedure:

1. Folin – Ciocalteau (Lowry) Assay for Protein Estimation:

1. Take ten tubes and label them as Blank and 1 to 9.

2. Make dilutions of Protein (BSA) standards with concentrations of 10, 20, 40, 80, 120, 160, 200 μg/200 µl by transferring respective amount of BSA from the standard protein solution (1 mg/ml) and adjusting it to a total volume of 200 μl by adding distilled water as mentioned in table 4.

3. Add 3 ml of Alkaline Copper reagent to each test tube including the Blank and Unknown tubes. Mix well.

4. Keep at room temperature for 10 minutes.

5. Add 0.5 ml of 1N Folin’s reagent to each test tube. Vortex the tubes and keep in Boiling Water Bath for 10 minutes

6. Switch on the Spectrophotometer, select the wavelength at 750 nm and let it warm before taking the absorbance (OD). First take the OD of Blank and make it zero

7. Remove Blank tube and take the OD of all the tubes and record it. Wash the cuvette after taking OD of each sample.

8. Plot a Standard Curve of absorbance at 750 nm on “Y” axis versus concentration of protein μg/200 μl on “X” axis.

9. Record the value “x” of Unknown from graph corresponding to the optical density reading of the test sample.

2.Bradford Assay for Protein Estimation:

1. Take eight tubes and label them as Blank and 1 to 7.

2. Make dilutions of Protein (BSA) standards with concentrations of 4, 8, 12, 16, 20 μg/200 μl by transferring respective amount of BSA from the standard protein solution (1mg/ml) and adjusting it to a total volume of 200 μl by adding distilled water as mentioned in table 6.

3. Add 1ml of Bradford’s reagent to each test tube including the Blank and Unknown tubes. Mix the contents of each tube thoroughly by vortexing the tubes and incubate at RT for 10 minutes.

4. Switch on the Spectrophotometer, select the wavelength at 595 nm and let it warm before taking the absorbance (OD). First take the OD of Blank and make it zero.

5. Remove Blank tube and take the OD of all the tubes within one hour and record it. Wash the cuvette after taking OD of each sample.

6. Plot a Standard Curve of absorbance at 595 nm on “Y” axis versus concentration of protein μg/200 μl on “X” axis.

7. Record the value “x” of Unknown from graph corresponding to the optical density reading of the test sample.

Calculation:

1. Folin – Ciocalteau (Lowry) Assay for Protein Estimation.

Determination of Protein Concentration in Unknown Sample:

Protein concentration can be calculated using the following formula:

Concentration of Unknown in “μg”

Protein Concentration in Test Sample = ------------------------------------------ x 5

Volume of sample in “μl”

= ___ µg/ml

2.Bradford Assay for Protein Estimation.

Protein concentration can be calculated using the following formula:

Concentration of Unknown in “μg”

Protein Concentration in Test Sample = -------------------------------------------- x 5

Volume of sample in “μl”

=____ µg/ml.

Observation & Result:

Interpretation:

1. Folin – Ciocalteau (Lowry) Assay for Protein Estimation.

The Lowry’s method of protein estimation is carried out by preparing a set of solutions with known protein concentrations and mixing them with the Folin’s reagent. A standard curve can be made and the concentrations of unknown protein sample can be derived from the standard curve.

2.Bradford Assay for Protein Estimation.

The Bradford’s method of protein Estimation is carried out by preparing a set of solutions with known protein concentrations and mixing them with the Bradford reagent. A standard curve can be made and the concentrations of unknown protein sample can be derived from the standard curve.

Questions:

1. what is meant by stock and working solution. explain by giving eg?

2. which method is more used for protein estimation and why ?

Reference Material

1. https://web.itu.edu.tr/~dulekgurgen/Proteins.pdf

2. https://fac.ksu.edu.sa/sites/default/files/lab_5_detection_and_quantitative_estimation_of_proteins_by_different_methods.pdf

3. https://www.youtube.com/watch?v=iNmmkZXgbf0

4. https://www.youtube.com/watch?v=Dh45qGrgw2g

5. https://www.youtube.com/watch?v=hdb3s4YHkms

Questions