CLINICAL CHEMISTRY (LABORATORY)

AUF | A.Y. 2023-2024

EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

PRE-DISCUSSION • It buffers pH, considered as a negative acute-phase reactant

TOTAL PROTEIN DETERMINATION protein and transports various substances in the blood.
Introduction • Measurement of albumin aids mainly in the diagnosis of liver
• Measurement of total protein is important in the assessment diseases. However, it can also be used in cases of
of different disorders. malnutrition and assessment of gastrointestinal, and kidney
• Since most proteins are synthesized in the liver, functions.
measurement of this analyte is especially useful in the
diagnosis of liver problems. Working Principle
• Impaired kidney function, intestinal malabsorption, • Albumin is bound by the bromcresol green dye to produce a
nutritional deficiency and dehydration are other health blue-green color that is measured at 630 nm. The color
conditions that may warrant the determination of total intensity produced is directly proportional to the
protein. concentration of albumin in the serum.

Working Principle Assay Requirement

• Protein in serum forms a violet colored complex when • Specimen: Serum
reacted with cupric ions in an alkaline solution. The intensity o Separate the serum from the cellular components
of the violet color is proportional to the amount of protein within 30 minutes of collection.
present when compared to a solution with known protein • Spectrophotometer Wavelength: 630 nm
concentration. • Temperature: 18-25°C
• Blank: Reagent Blank
Assay Requirement • Reagent Component:
• Specimen: Serum o Bromocresol Green (BCG)
o Separate the serum from the cellular components o Buffer, pH 4.6
within 30 minutes of collection. o Surfactant
• Spectrophotometer Wavelength: 540 nm o Stabilizers
• Temperature: 37°C
• Blank: Reagent Blank Assay Procedure
• Reagent Component: • Mix and incubate the tubes at room temperature (18-25°C)
o Sodium Hydroxide for 1 minute
o Copper Sulfate • Read the absorbance of the standard and sample against a
o Sodium Potassium Tartrate reagent blank within 60 minutes
o Potassium Iodide • Compute for the level (concentration) of albumin in the
sample
Assay Procedure • Calculation (with standard):
• Let the tubes stand at room temperature (18-25°C) for 5
minutes
• Reaad the absorbance of the standard and sample against a
reagent blank within 60 minutes. • Concentration of the standard: 4 g/dL
• Compute for the level (concentration) of total protein in the • Reference range (as per manufacturer):
sample o 3.5 – 5.3 g/dL
• Calculation (with standard):
POST-DISCUSSION
TOTAL PROTEIN MEASUREMENT

• Concentration of the standard: 8 g/dL

• Reference range (as per manufacturer):
o 6.2 – 8.5 g/dL

ALBUMIN DETERMINATION
Introduction
• Albumin, the most abundant type of protein in the plasma, is
synthesized in the liver. It is responsible for the colloid
osmotic pressure of the intravascular fluid.

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 CLINICAL CHEMISTRY (LABORATORY)
AUF | A.Y. 2023-2024
EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

Kjeldahl Method o Amido Black B

• Principle: Protein nitrogen is converted to NH4 by heating o Lissamine Green
with Sulfuric acid in the presence of a catalyst. o Coomassie Brilliant Blue
• Protein precipitation step may be required
o Organic acids (e.g., TCA, Tungstic Acid) Interferences or Potential Causes of Error
o NPN’s removed with the supernatant • Unequal dye-binding response of individual proteins.
• Protein Digestion • Difficult calibration.
o H2SO4 plus Δ (340-360 Celsius) • Pyrogallol Red
o Purpose: oxidizes C,H, S → CO2, CO, H2O, SO2 o One of the most commonly used dyes for urine and
thereby leaving Nitrogen only in the solution CSF protein analysis.
• Nitrogen Conversion into Ammonium Bisulfite (NH4, HSO4)
o Alkalinization ALBUMIN METHODS
o Distillation
o Acid Titration
• The whole point is to extract Nitrogen from the specimen.
• sometimes used as a reference method (Tietz, et al. 2018).
• 1° assumptions:
o Proteins contains approx. 16% Nitrogen
o No clinically significant proteins are lost in the
precipitation step.
• One of the first reproducible method used for total protein
measurement
• Time consuming (impractical for routine use)

Biuret Method Dye-Binding Albumin Procedure

• Principle: under strong alkaline conditions, Cu2+ ions form • Positive interference for Methyl Orange:
multivalent complexes with peptide bonds in proteins. o β-lipoproteins, α1 and α2-globulins
o This binding shifts the absorption maxima of Cu2+ • Negative interference with HABA:
from blue to violet-colored chelate. This is called o Salicylates, penicillin, B2, sulfonamides (competes
“The Biuret Reaction”. with Albumin for HABA - falsely low Albumin
• Reagents: values).
o Sodium Potassium Tartrate (Stabilizer for Cu2+) • Positive interference with BCG:
o KI2 (Antioxidant) o 100mg/dL cell-free Hemoglobin results in 0.1g/dL
• Read at 540nm. increase in Alb.
o The resultant absorbance is directly proportional to o Nephrotic syndrome, End-stage Renal Disease-
the number of peptide bonds present in the Elevated α-globulin fractions (Utility of BCG can
solution. (Endpoint Biuret Assay). result to an overestimation of a true low Albumin
• Lipemia is considered as an interferent. value [i.e., false increase]).
• Specimen rich in Proline results in reduced reactivity. • Negative Interference with BCP:
• At least 2 peptide bonds must be present (for the generation o Structurally altered Albumin or Albumin-attached
of stable chelate) molecules inhibits Albumin-BCP binding.
• Rate Biuret Assay
SERUM PROTEIN ELECTROPHORESIS
o Additional absorbance change depends on the rate
of unfolding of a protein and exposure of additional • SPE relies on the separation of proteins based on their net
binding sites for Cu2+ ions under strongly alkaline electrical charges, size, properties of the support medium,
conditions. and temperature of operation.
• Semi-quantitative method.
Dye-Binding Methods • Principle:
• Most serum proteins binds to dye. Protein-dye complex o When an electric field is applied to a medium
shifts the absorbance spectra of dyes which can be containing charged particles:
measured using a spectrophotometer. o The negatively charged particles migrate toward the
• Commonly used dyes: positive electrode (anode)
o Bromphenol Blue o The positively charged particles migrate toward the
o Ponceau S cathode.

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 CLINICAL CHEMISTRY (LABORATORY)
AUF | A.Y. 2023-2024
EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

o At pH 8.6, most serum proteins have a negative b. The isoelectric point of protein is the pH at which a
charge. This separates the protein fractions in protein has no net charge.
serum when an electrical field is applied. c. At pH 8.6, proteins are negatively charged and
• On the support medium (cellulose acetate or agarose gel), migrate toward the anode.
the pattern of migration is as follows: d. If the buffer pH is higher than the isoelectric point
o Albumin is most anodic (because of its small size of protein, the protein carries a negative charge and
and large number of negative charges), then α1- migrates toward the anode.
globulins, α2-globulins, β-globulins; λ-globulins are 2. Connect support medium to two electrodes and let the
most cathodic. current pass through the medium
a. ALL major proteins at this point carries a net
negative charge, when current is applied these
proteins have no other choice but to migrate
towards the positive terminal (i.e., the anode).
b. as every other observable phenomena, what’s left
now is all the other variables that can influence the
RATE of these protein’s MIGRATION

MIGRATION RATE
• Charge of the molecule - which is directly proportional to
rate of movement
• Size of the molecule - which is inversely proportional to
rate of movement
• Electrical field - which increased current increases
migration rate
• Ionic strength of buffer - which increased ionic strength
decreases migration rate
• pH of buffer - which decreased pH slows migration
• Viscosity of supporting medium - which is inversely
proportional to migration rate
• System temperature - which high temperature can
denature protein and slow migration

3. After separation, fix the supporting medium in an acid

solution.
a. Acetic acid denatures, thereby immobilizing the
proteins
4. Stain
a. In order to visualize the bands, the proteins now
currently suspended in the medium is stained by
dyes used in Dye-binding procedure(i.e., Ponceau
S, Amido Black B, CBB and so on).
b. By this time, you have this in your arsenal

This is a strip-chart record generated by a scanning densitometer

STEPS IN SERUM PROTEIN ELECTROPHORESIS

1. Apply serum specimen close to the cathodic end of the
support medium
a. Proteins are amphoteric (i.e., they can have positive
or negative charge because of their acidic and basic
side chains).

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 CLINICAL CHEMISTRY (LABORATORY)
AUF | A.Y. 2023-2024
EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

5. Place the fixed and stained medium in a scanning CLINICAL CORRELATIONS

densitometer.
a. The electrophoretic pattern you now have
suspended in the medium(i.e., cellulose acetate,
agarose or PAGE) is colored, hence they can
TRANSMIT light.
b. Inside a densitometer, light is transmitted through
the membrane and a phototube records the
absorbance of the protein-dye complex present in
each of the protein fraction you have just stained.
6. Read/interpret the strip-chart based on the pattern of the
fractions and your knowledge of protein met and
pathophysiology.

a. Scanning densitometer reports the percentage of

the total dye that appears in each fractions.
b. The concentration is then calculated as a
percentage of the total protein determined by one
of the total protein methods(i.e., Biuret etc).

REFERENCE RANGES FOR THE MAJOR PROTEIN FRACTIONS

A reference serum control is processed with each
electrophoretic run.
• Albumin, 53% to 65% of the total protein (3.5 to 5.0
g/dL)
• α1-Globulin, 2.5% to 5% (0.1 to 0.3 g/dL.)
• α2-Globulin, 7% to 13% (0.6 to 1.0 g/dL.)
• ß-Globulin, 8% to 14% (0.7 to 1.1 g/dL.)
• γ-Globulin, 12% to 22% (0.8 to 1.6 g/dL)

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 CLINICAL CHEMISTRY (LABORATORY)
AUF | A.Y. 2023-2024
EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

HIGH RESOLUTION PROTEIN ELECTROPHORESIS

• Provides protein fractionation up to 12 bands
• Some unique features:
o Higher voltage
o cooling system
o more concentrated buffer
• Agarose gel is the most commonly used medium
• Same steps as with the usual SPE is followed when
performing HRE
• Particularly useful in:
o detecting small monoclonal bands
o differentiating unusual bands or prominent
increase of normal bands that can be confused
with monoclonal gammopathy
• Remember that the most significant finding from an
electrophoretic pattern is the ID of monoclonal
immunoglobulin disease
o >>HRE comes in to play for a more definitive
approach from this fact.

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 CLINICAL CHEMISTRY (LABORATORY)
AUF | A.Y. 2023-2024
EXERCISE 11-12: LABORATORY IDENTIFICATION OF PROTEINS

ANCILLARY TECHNIQUES IMMUNOFIXATION ELECTROPHORESIS

Capillary Electrophoresis • Qualitative method for evaluation of immunoglobulins.
• Electrophoresis are carried out in a small-bore, fused silica • Proteins are electrophoresed into five zones as in SPE. Then,
capillary tube. monospecific antiserum is added, and the support medium
• This capillary tube serves as an electrophoresis chamber is stained for visual interpretation of bands.
that is connected to a detector at its terminal end and to a • It is used to analyze protein concentration in serum, urine,
power supply. and other fluids.
• Buffers include Tris, borate, acetate, formate, and
phosphate.
• Simplest form of electrophoresis
• It is able to resolve many analytes such as proteins, peptides,
and amino acids.

Isoelectric Focusing
• Allows amphoteric molecules such as proteins to be
separated by electrophoresis in a pH gradient generated
between the cathode and anode.
• A solute migrates to a point where its net charge is zero
(isoelectric point).
• At the solute’s isoelectric point (pi), migration stops, and the
sample is focused into a tight zone.
• This technique is commonly employed in protein
characterization to determine a protein's pl.

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