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Cch10 Protein2
Cch10 Protein2
Cch10 Protein2
PROTEINS
Objective
• simple proteins
• complex proteins :
- apoproteins,
- conjugated proteins
Protein classification, continued….
Albumin
• the most abundant plasma protein extra vascular
body fluids, including CSF, Interstitial fluid, urine,
and amniotic fluid.
• accounts approximately one-half of the plasma
protein mass.
• a globular protein, with molecular mass of 66.3
KD.
• Because of its high net negative charge at
physiological pH, highly soluble in water, but
does not have carbohydrate side chain.
Functions of albumin
• Molecular size
• Differential solubility
• Electrical charge
• Adsorption on finely divided inert materials
• Specific binding to antibodies, coenzymes, or
hormone receptors
Specific methods for total protein determination.
• Biuret method
• Direct photometric methods.
• Dye-binding methods.
• Turbidimetric and nephelometric methods
Biuret method
Dye-binding methods
Salt fractionation or the 'salting-out'
procedure
By difference
Electrophoresis
Immunochemical techniques.
Dye binding method
BCG Method
Test principle: Albumin and BCG are allowed to bind
at pH 4.2, in succinate buffer,
absorption of the BCG-albumin complex is measured
at 628 nm.
At pH 4.2, albumin acts as a cation to bind the
anionic dye.
The reaction is extremely fast and goes to
completion in only a few seconds.
Reference Range
hyperlipemia
hyperbilirubinemia
hemolysis
can generally be eliminated (minimized) by
dilution of serum 1:250
BCP Method
Test principle:
Yellow BCP dye, buffered at pH 5.2 with
acetate
turns green when complexed with albumin.
Absorbance of the green complex is
measured at 603 nm.
Specimen
serum is recommended
Results tend to be erroneous if the overall
serum protein pattern is abnormal
Methods for the determination of total globulins
Test principle:
glyoxylic acid reacts with tryptophan residues
of proteins to form a purple color.
Copper sulphate is added to enhance color
formation.
human globulins are known to contain 2 - 3%
tryptophan
Serum Protein Electrophoresis
• Electrophoresis is widely used in clinical
laboratories to study and measure the protein
content of biological fluids- serum, urine or csf.
• Screening tool for prtein abnormalities
• Electrophoresis techniques include:
– Cellulose acetate electrophoresis
– Gel and capillary electrophoresis
– Specialized techniques termed western
blotting, immunofixation, and two-dimensional
electrophoresis
Methodology for Protein Electrophoresis
Patient’s specimen is placed into a sample
trough within agarose gel, is placed in an
alkaline buffer solution
a standardized voltage is applied and
allowed to run for 1hr
the agarose gel is processed in acetic
acid and alcohol washes to fix the
proteins in the agarose.
the protein fractions are stained with
Coomassie Brilliant Blue protein stain.
After a second wash, fixed protein bands
can be visualized and quantified with
densitometry.
In normal serum electrophoresis 5-6
bands are visible:
Albumin
Globulins: α1-, α2-, β-, and γ-
Materials and procedures of protein
electrophoresis
Albumin 1 2
+ -
Specimen for electrophoresis
• Serum
• CSF
• Concentrated urine
Interpretation of Results
Reference Range of total protein
– Serum---------------------------6-8 g/dl
– CSF----------------------------- 8-32 mg/dl
For electrophoresis
-serum: albumin-----------------3.9-5.1 g/dl
α1-globulin------------0.2-0.4 g/dl
α2-globulin------------0.4-0.8 g/dl
β-globulin--------------0.5-1.0 g/dl
γ-globulin---------------0.6-1.3 g/d
Compare the patient results with the reference range to
assess for hyper- or hypoglycemia
Quality Control
A normal & abnormal quality control sample should be
analyzed along with patient samples, using Westgard or
other quality control rules for acceptance or rejection of
the analytical run.
– Assayed known samples
– Commercially manufactured
Validate patient results