DIAGNOSTIC IN CLINICAL CHEMISTRY

I
MKEB 2404
DATE:
24 JANUARI 2007
TITLE:
• MEASUREMENT OF TOTAL PROTEIN (BIURET METHOD)
AIMS & OBJECTIVE:
• To learn one of the common method for protein estimation by using photometric
colorimetric test.
• To learn how to use a calibration curve for the determination of an unknown
concentration of a sample.
PRINCIPLE:
• Cupric ions react with protein in alkaline solution to form a purple complex.
• The absorbance of this complex is proportional to the protein concentration in
the sample.
PROCEDURE:
1. You are provided with Bovine Serum Albumin (BSA) standard that is 100
mg/ml.
2. Set up a series of dilution and assay for protein using the Biuret method (You are
to plan your own series of dilution.).
3. Follow the assay protocol given. Take the absorbance for each dilution.
4. Plot absorbance versus concentration.
5. Find the concentration of protein in the sample.
ASSAY PROTOCOL
• Wavelength: 540 nm
• Measurement: Against reagent blank.
• Only one reagent blank per series is required.

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PIPETTING SCHEME

Sample/ Standard/
Reagent blank
Control
Distilled water 20 ul -
Sample or Standard or - 20 ul
Color reagent
Control 1000 ul 1000 ul
Mix, incubate for 5 min at 20-25 °C. Measure the absorbance of the sample and the
standard against the reagent blank within 60 min.

RESULTS & CALCULATION:

TUBES CALCULATION ABSORBANCE
STANDARD: • Total Volume : 200
• 20 mg/dL ul
• M1V1 : M2V2
○ 100(V1) =
20(200) 0.066
○ V1 =
200(20)/100
= 40 ul
• H2O = 160 ul
• 40 mg/dL • Total Volume : 200
ul
• M1V1 : M2V2
○ 100(V1) =
40(200) 0.147
○ V1 =
200(40)/100
= 80 ul
H2O = 120 ul
• 60 mg/dL • Total Volume : 200 0.229
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 ul
• M1V1 : M2V2
○ 100(V1) =
60(200)
○ V1 =
200(60)/100
= 120 ul
• H2O = 80 ul
• 80 mg/dL • Total Volume : 200
ul
• M1V1 : M2V2
○ 100(V1) =
80(200) 0.310
○ V1 =
200(80)/100
= 160 ul
• H2O = 40 ul
• 100 mg/d • Total Volume : 200
ul
• M1V1 : M2V2
○ 100(V1) =
100(200) 0.391
○ V1 =
200(100)/100
= 200 ul
• H2O = 0 ul
Control - 0.289
Sample A - 0.290
Sample B - 0.292

CONTROL RANGE:
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 • 76.27 mg/dL +/- 1.659 (2SD)

RESULT FROM GRAPH LINE:

RESULT
Control 71.0 mg/dL
Sample A 72.0 mg/dL
Sample B 73.0 mg/dL

DISCUSSION:
1. Explain how you do the dilutions of standard.
The dilution process is shown in table below:
Volume Of Total Volume
Concentration Volume Of
Tube Distilled Water (μL) for each
(mg/dL) BSA (ml)
(μL)) tube
1 20 40 160 200
2 40 80 120 200
3 60 120 80 200
4 80 160 40 200
5 100 200 0 200

2. Discuss the other methods of protein analysis.

Method Principle Comment
kjeldahl • Digestion of • Reference method;
protein; assume average
measurement of nitrogen content of
nitrogen content. 16%
Refractometry • Measurement of • Rapid and simple;
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 refractive index assume non protein
due to solutes in solids are present in
serum. same concentration as
in the calibrating
serum.
Biuret • Formation of • Routine method;
violet-colored requires at least two
between Cu ions peptide bonds and an
and peptide alkaline medium.
bonds.
Dye-binding • Protein binds to • For research purpose
dye and causes a
spectral shift in
the absorbance
maximum of the
cycle.
Ultraviolet absoption • The absorptivity • Rarely used in clinical
is related to the laboratory.
absorbance of • Mostly in research
tyrosine, purpose.
trytophan, and
phenyl-alanine
amino acids in
the protein.
Lowry • Combination of • Rarely used in clinical
Biuret reagent laboratory.
with folin- • Mostly in research
Ciocalteu reagent purpose.
where the
reaction is
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 measured by the
absorbance.
Bradford • Complex color
formed due to
reaction of
protein with
Coomasie
Brilliant Blue dye.

3. Interference in Biuret Method.

METHOD INTERFERENCES
1. The reagent is expired.
2. The serum is hemolysed.
3. Pipetting error.
4. Presence of abnormally small protein,
such seen in multiple myeloma.
5. Delaying the serum analysis by leave it
BIURET
in room temperature for long time.
6. Forming a precipitation. (Sodium
potassium to complex tartrate).
7. No color reaction is formed.
8. More reactive compound is added
such as cupric ions.

CONCLUSIONS:
1. There are many methods to measure total protein.
2. But, on this experiment the Biuret method is used for determination the protein.
3. Finally, the result that obtained from this experiment are;
• Control : 71.0 mg/dL

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• Sample A : 72.0 mg/dL
• Sample B : 73.0 mg/dL

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