Biochemistry - Laboratory 1º Veterinaria 18-19

Laboratory work nº 1: Quantitative proteins estimation by Lowry method
DEPARTAMENTO DE BIOQUíMICA Y BIOLOGÍA MOLECULAR

Universidad de Córdoba
Grado de Veterinaria. 2018/2019
Biochemistry. Laboratory work.
Laboratory work nº 1
Quantitative proteins estimation by Lowry method
INTRODUCTION
The estimation of the amount of protein in a given sample is a basic, usual and essential
technique in the clinical laboratories and in the industry.
Several colorimetric methods are commonly used for determination of protein concentration:
Direct Methods: The proteins absorb UV and IR lights:
v Absorbance at 280 nm (Layne., 1957): Depends on the presence of aromatic amino acids
in proteins (Trp, Try and Phe).
v Absorbance at 210-225 nm (Stoscheck.,1990)
v Absorbance at ~ 1650 nm (amida I) and ~ 1550 nm (amida II)
Indirect Methods: Relative method. This methods need a standard curve with known
amounts of purified protein.
Protein-dye complex Change of the absorption spectrum maximum
v Bradford, Coomasie Brilliant Blue G-250, (Bradford.,1976)
Protein-sustrate reaction Colorimetric detection of products
v Biuret (Gornal et al.,1949)

v Lowry (Lowry et al.,1951)
v BCA, bicinchoninic acid (Smith et al.,1985)
All protein assays have advantages and disadvantages, with a wide range of options for
accurate protein concentration determination based on assay time, sensitivity, specificity of the
method, interference by individual substances or on the capability to recuperate the sample
after the assay.
Currently, clinical laboratories use specific kits and new equipment to carry out massive
analysis of samples. To carry out these assays you need to be familiar with the equipment
without other acknowledgment. However, a Biochemistry student has to know how the assay
works, as well as how to process the assay.
OBJECTIVE
The student needs to be familiar with one of the methods used to determine protein
concentration (Lowry method) and how to calculate the concentration of a solution and dilutions
numbers.
Biochemistry. Grado en Veterinaria. Curso 1º 1
THEORETICAL BASIS
Lowry’s procedure is one of the most accurate and sensitive method of determining protein
concentration.
The method is based on the combination of Biuret reaction with Folin-Ciocalteau reaction.
The reactions result in a strong blue color, which depends partly on the tyrosyne and tryptophan
content (Absorbance between 600 and 800 nm).
1) The first reaction is the reduction of copper ions (Cu2+) under alkaline conditions, which
forms a complex with peptide bonds. The folding of the protein exposes the phenolic Tyr
residues, which will participate in the second reaction. This first reaction, Biuret, is not
sensitive enough, however the second reaction increase the sensitivity of the assay.
2) The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond

complex, which subsequently causes a colour change of the solution into blue with an
absorption in the range of 650 to 750 nm, detectable with a spectrophotometer. The
colour intensity is directly proportional to the protein concentration.
The protein concentration in a given sample is determined by the comparison with a standard
curve with known amounts of purified protein, bovine serum albumin (BSA). Each group will
prepare a standard curve to determine the protein concentration in the sample.

MATERIALS
• Distilled water: in a plastic tube.

• BSA solution (0.25 mg/mL): in eppendorf tube.
• Unknown diluted protein sample: in eppendorf tube.
• Reagent I (prepare just on time before use):
Reagents:
o 2% Na2CO3 in 0,1 N NaOH: in a plastic tube.
o 4% Potassium sodium tartrate: in eppendorf tube.
o 2% CuSO4 · 5 H2O: in eppendorf tube.
Procedure to prepare the Reagent I: take 9.8 mL of 2% Na2CO3 in 0,1 N NaOH with
a glass pipette and drop it in a test tube; add 100 µL of 4% potassium sodium tartrate
and 100 µL of 2% CuSO4 · 5 H2O and mix well by pipetting up and down multiple
times; The product should be a transparent and bluish solution.
• Reagent II (Folin-Ciocalteau Reagent): 1/3 Dilution of the comercial reagent with distilled
water (the solution is already diluted in the eppendorf and ready to use).
• 1 test tube and eppendorf tubes.
• Racks for test and eppendorf tubes
• 10 mL Glass pipette and 1000 y 200 µL micropipettes with the pipet tips required.
• Water bath at 45 °C.
• Colorimetry fixed at 650 nm.
PROTOCOL
To make the standard curve and determine the protein concentration of the sample, you
should follow the instructions given in the below table:
Test tubes and µL of the reagents per tubo

Reagents 0 1 2 3 4 5 Sample
Distilled water 200 160 140 100 60 ––– –––
BSA Sol. (0.25 mg/mL) ––– 40 60 100 140 200 –––
Sample ––– ––– ––– ––– ––– ––– 200
Reagent I 720 720 720 720 720 720 720
Mix with vortex and incubate for 10 minutes at 45 ºC
Reagent II (Folin 1/3) 80 80 80 80 80 80 80
Mix with vortex and incubate for 15 minutes at RT (room temperature)
Record absorbance values at 650 nm
We will use only one plastic cuvette for all the measurements. Firstly, we will calibrate with
the zero absorbance (0 test tube). For the rest of the measurements, we will measure from the
lower (light colour) to the higher concentration (strong colour).

Using the initial BSA concentration, calculate the BSA concentration (g/dL) in each test tube
(1 to 5), taking into account the dilution factor.
Plot the standard curve on the graph paper (protein concentration (g/dL) in abscissa and A650
in ordinate). The protein concentration in the sample tube will be determined by inserting the
A650 in the standard curve.To calculate the final protein concentration you should take the
dilution factor into account.
Once the students finish the experimental procedure, they will analyze the results in next
page. The students will deliver the results at the end of the class.

SURNAME, NAME ……………………………… DNI:………………… Group:……
DETERMINATION OF TOTAL PROTEIN CONCENTRATION
1) Graph the standard curve: Absorbance values (A ) versus protein concentration (g/dL)
650
2) Calculate the total protein concentration (g/dL) of the given sample.
Tube Vol BSA (mL) BSA (g/dL) A

650
1
2
3
4
5
Sample

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Laboratory work nº 1: Quantitative proteins estimation by Lowry method

DEPARTAMENTO DE BIOQUíMICA Y BIOLOGÍA MOLECULAR

Direct Methods: The proteins absorb UV and IR lights:

Protein-dye complex Change of the absorption spectrum maximum

v Bradford, Coomasie Brilliant Blue G-250, (Bradford.,1976)

Protein-sustrate reaction Colorimetric detection of products

v Biuret (Gornal et al.,1949)

2) The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond

Biochemistry. Grado en Veterinaria. Curso 1º 2

• Distilled water: in a plastic tube.

Test tubes and µL of the reagents per tubo

Biochemistry. Grado en Veterinaria. Curso 1º 3

Biochemistry. Grado en Veterinaria. Curso 1º 4

SURNAME, NAME ……………………………… DNI:………………… Group:……

DETERMINATION OF TOTAL PROTEIN CONCENTRATION

2) Calculate the total protein concentration (g/dL) of the given sample.

Tube Vol BSA (mL) BSA (g/dL) A

Biochemistry. Grado en Veterinaria. Curso 1º 5

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