The document discusses plasma proteins, including their definition, functions, compositions, detection methods, and electrophoresis. Plasma proteins comprise 7% of plasma solids and include albumin, globulins, fibrinogen, and others. They serve important roles like nutrient transport, coagulation, and osmotic balance. Detection methods discussed are precipitation, electrophoresis, immunoassays, and more. Serum protein electrophoresis separates proteins by size and charge and is commonly used to analyze plasma protein composition.
2. Contents
Definition of plasma proteins
Functions
Compositions
Follow-up plasma protein test
Methods for detection of different plasma proteins
Protein electrophoresis
References
3. Plasma Proteins
The proteins present in the plasma of human blood are a mixture of simple proteins, glycoproteins,
lipoproteins and other conjugated proteins are called “Plasma Proteins“. These may be separated by salt
precipitation, immunological technique and electrophoresis
• Plasma is obtained from anti-coagulated blood
• Plasma proteins forms 7% of the solids in plasma
• Total protein content of normal plasma is 6-8g/100ml
• Almost all plasma proteins are synthesized by liver except immunoglobulin
4. Functions
Protein nutrition
Osmotic pressure and water balance
Buffering action
Transport of lipids
Transport of other substances
Blood coagulation
8. Further Composition of Plasma Proteins
• Transferrin
• Hemopexin
• Low density lipoprotein LDL
• β2 macroglobulin
• C4 complement
• C3 compliment
• C1q complement
• C-reactive protein
β-globulin 13.4%
9. Further Composition of Plasma Proteins
• Ig G
• Ig M
• Ig E
• Ig A
• Ig D
γ-globulin 11.0%
10. Follow Up Plasma Protein Test
• C-reactive protein tests to evaluate for inflammation
• Immunoglobulin tests to measure antibodies
• liver enzyme tests
• Protein electrophoresis to look for underlying bone marrow disorders
11. Methods for detection of Plasma Protein
• Precipitation method
• BCG method
• electrophoresis
• Ultra centrifugation
• Precipitation method
• Radioimmunoassay
• Enzyme-labelled Immunoassay
• Serum protein electrophoresis
Albumin
For α1-fetoprotein (AFP)
α1-Globulin
12. Methods for detection of Plasma Protein
• Precipitation method
• Serum protein electrophoresis
• Immunodiffusion
• Immunonephelometric assay
• Immunofixation
• Radial immunodiffusion
• Immunoturbidity
• Nephelometry
• Immunofixation
For α1-anti trypsin
For α1-acid glycoprotein
13. Methods for detection of Plasma Protein
• Radial immunodiffusion
• Immunonephelometric
• Radial immunodiffusion
• Immunonephelometric
For Hepatoglobin
α2-Globulin
For ceruloplasmin
• Radial immunodiffusion
• Immunonephelometric
• ELISA
• Latex agglutination
immunoassay
For α2-microglobin
14. Methods for detection of Plasma Protein
• Radial immunodiffusion
• Immunonephelometric
• TIBC
• Radial immunodiffusion
• ELISA
• Nephelometry
• CRP assay
For Transferrin β-Globulin
For Hemopexin
• Immunoassay
• Serum protein
electrophoresis
• Precipitation
• Nephelometric
immunoassay
• Turbidimetry
For β2-microglobin
For LDL
For ComplementCRP
15. Methods for detection of Plasma Protein
• Radial immunodiffusion
• Nephelometry
• Turbidimetry
• Electro chemiluminescent immunoassay
• Radioimmunoassay
• Screening
• Serum protein electrophoresis
γ-Globulin
16. Methods for detection of Plasma Protein
• Radial immunodiffusion
• Nephelometry
• Turbidimetry
• Radioimmunoassay
• Plasma protein electrophoresis
• Clotting time CT
Fibrinogen
17. Principles of Methods for detection of
Plasma Protein
Radial immunodiffusion
An antigen sample is placed in a well and
allowed to diffuse into agar containing a
suitable dilution of an antiserum.
As the antigen diffuses into the agar, the
region of equivalence is established and a
ring of precipitation, a precipitin ring, forms
around the well.
The area of the precipitin ring is
proportional to the concentration of
antigen.
.
Nephelometry
The principle is to measure forwarded scattered light when a laser beam passes through a sample and the light is deflected by
the particles.
Radioimmunoassay
The principle of RIA involves competitive binding of
radiolabeled antigen and unlabeled antigen to a high-affinity
antibody.
The labeled antigen is mixed with antibody at a concentration
that saturates the antigen-binding sites of the antibody.
Then test samples of unlabeled antigen of unknown
concentration are added in progressively larger amounts
The more unlabeled antigen is present, the less radioactivity there
is in the complex.
The concentration of the unknown (unlabeled) antigen or hapten
is determined by comparison with the effect of standards.
18. Plasma Protein Electrophoresis
Electrophoresis is a separations technique that is based on the mobility of ions in an electric field. Ions
have different migration rates depending on their total charge, size, and shape, and can therefore be
separated. The technique is used particularly for macromolecules, such as proteins.
• Gel electrophoresis involves the use of gel as supporting media for separation of DNA, RNA or
proteins under the influence of electric charge. It is usually performed for analytical purposes but may
be used as a preparative technique to partially purify molecules prior to use for other methods such as
mass spectrometry, PCR, cloning, DNA sequencing and immuno-blotting.
• This is the most commonly used electrophoresis in biotechnology laboratories
19. Plasma Protein Electrophoresis
It may be of two types;
1. Agarose gel electrophoresis
Mostly for the separation of DNA fragments having more than 50 base pairs
2. PAGE (polyacrylamide gel electrophoresis)
For separation of proteins
It may be Sodium Dodecyl Sulfate SDS-PAGE and NATIVE
20. Sodium Dodecyl Sulfate (SDS)
polyacrylamide gel electrophoresis is mostly
used to separate proteins accordingly by size.
This is one of the most powerful techniques to
separate proteins on the basis of their molecular
weight.
Principle
This technique uses anionic detergent Sodium Dodecyl
Sulfate (SDS) which dissociates proteins into their
individual polypeptide subunits and gives a uniform
negative charge along each denatured polypeptide.
Power Supply:
A power supply of 100-200 volts is
needed. This is ideal for running and
transferring protein resolving gels.
Buffer:
Two types of buffers are used in SDS-PAGE. The
lower reservoir (which has the running gel) has
amine buffers. It is adjusted by using HCl. The
upper reservoir (which has stacking gel) also has
amine buffers but its pH is slightly above that of
running gel buffer and is adjusted with glycine
instead of HCl.Stain
Coomassie Brilliant Blue R-250 (CBB)
is the most popular protein stain (blue
bands).
2- SDS-PAGE
Reader
Densitometric scanning machine
