Albumin: Conc. 60%, M.W. 69000, 585 AAs with 17 disulphide
1. Synthesized from liver.
2. Maintains colloidal osmotic pressure.
3. Decreasing causes edema.
4. Serves as asource of AA
5. Decresed Alb –cirrhosis,nephrotic syndrome, malnutrition.
6. Increased alb - dehydration
1. It’s a glycoprotein with m.w. 90000 – 130000.
3. The α & β helps to transport proteins, hormones, vitamins, minerals
4. γ globulins functions as immunoglobulins.
Total proteins - 6-8 gm/dl (100%)
Alb - 3.5 – 5 gm/dl (60%)
Glob - 2.5 – 3 gm/dl (40%)
α1 - 3%
α2 - 11%
β - 11%
γ - 15%
METHODS FOR SEPARATION OF SERUM
1. Precipitation by salts.
2. Cohn’s fractional precipitation method.
3. Sedimentation by ultracentrifugation.
4. Paper chromatography.
Definition: Electrophoresis is the migration of charged
molecules in an electric field. The negative charged particles
(anions) moves towards positive charged electrodes (anode).
Positively charged particles (cations) moves towards cathod
(negatively charged electrode).
1. Depending upon the nature of supporting medium
a. Agar gel electrophoresis (AGE).
b. PAGE, SDS PAGE, QPNC PAGE (Quantitative preparative native
c. Cellulose acetate electrophoresis.
d. Capillary electrophoresis.
2. Depending upon the mode of technique.
a. Slide gel electrophoresis.
b. Tube gel electrophoresis.
c. Disc electrophoresis.
d. Low and high voltage electrophoresis.
e. Two dimensional gel electrophoresis
Applications of Electrophoresis:
1. Separating serum proteins for diagnostic purpose.
2. Haemoglobin separation.
3. Lipoprotein separation and identification.
4. Isoenzyme separation and their analysis.
5. Nucleic acid studies.
6. Determination of molecular weight of the proteins.
FACTORS AFFECTING ELECTROPHORESIS
I. The electric field:
a. Voltage - V ∞ M
b. Current - C ∞ M
c. Resistance- R 1/∞ M
II. The sample:
a. Charge - C ∞ M
b. Size - S 1/∞ M
c. Shape - Molecules of similar size but different shape such as fibrus
and globular proteins exhibit diffeent migration
characteristics. Because of the differential effect of
frictional and electrophoretic force.
III. The buffers:
This determines and stabilizes the pH of the supporting medium and hence affects
the migration rate of compound in a number of ways.
a. Composition: The buffer should be such that it does not binds with the compounds
to be separated as this may alters the rate of migration. Therefore barbitone buffer
is always preferred for the separation of proteins or lipoproteins.
b. Concentration: As the ionic strength of the buffThe er increases the proportion of
current carried by the buffer will increase and the share of the current carried by the
sample will decrease thus slowing down the rate of migration.
c. pH: For organic compounds pH determines the extent of ionization and therefore
degree and direction of migration are pH dependent.
d. The supporting medium: The composition of supporting medium may cause
adsorption, electro osmosis and molecular sieving. Which may influence the rate of
migration of compounds. The commonly using supporting medium in the
laboratory are agarose, polyacrylamide and cellulose acetate membrane.
Types of buffers used in electrophoresis
1. Tris buffer.
2. Glycine buffer.
3. Sodium barbituric acid.
4. TAE buffer (Tris acidic acid EDTA).
Types of StainsFor serum proteins – Amido block
- Coomassie brilliant blue
For isoenzymes- Nitro tetra zolium blue
For lipoprotein zones- Fat red 7B
- Oil red O
- Sudan block B
For DNA fragments - Ethidium bromide
For CSA proteins - Silver nitrate
What is needed?
Agarose - a
polysaccharide made from
seaweed. Agarose is
dissolved in buffer and
heated, then cools to a
gelatinous solid with a
network of crosslinked
Some gels are made with
acrylamide if sharper
bands are required
Buffer - in this case
The buffer provides
ions in solution to
Not only is the agarose
dissolved in buffer, but
the gel slab is
gel) in buffer after
Also needed are a
power supply and a gel
Gel chambers come in
a variety of models,
and a variety of sizes
A gel being run
DNA loaded in
wells in the agarose
To make loading wells easier
The comb is
little ‘wells’ and
buffer is poured over
the gel to cover it
The serum samples
are mixed with a
dense loading dye
so they sink into their
wells and can be
The serum samples
are put in the wells
with a micropipette.
disposable tips and
1/1,000,000 of a litre
Pulsed field gel electrophoresis
Pulsed Field Gel Electrophoresis (commonly
abbreviated as PFGE) is a method for separating large
DNA molecules, which may be used for genotyping or
Under normal electrophoresis, large nucleic acid
particles (above 30-50 kb) migrate at similar rates,
regardless of size. By changing the direction of the
electric field frequently, much greater size resolution
can be obtained
SDS-PAGE, officially sodium dodecyl sulfate
polyacrylamide gel electrophoresis, is a technique
used in biochemistry, genetics and molecular biology
to separate proteins according to their electrophoretic
Quantitative preparative native continuous
polyacrylamide gel electrophoresis (QPNC-PAGE) is a
new method for separating native metalloproteins in
complex biological matrices.
Indications for Serum Protein Electrophoresis
Suspected multiple myeloma, WaldenstrÃ¶m's macroglobulinemia, primary amyloidosis,
or related disorder
Unexplained peripheral neuropathy (not attributed to longstanding diabetes mellitus,
toxin exposure, chemotherapy, etc.)
New-onset anemia associated with renal failure or insufficiency and bone pain
Back pain in which multiple myeloma is suspected
Hypercalcemia attributed to possible malignancy (e.g., associated weight loss, fatigue,
bone pain, abnormal bleeding)
Rouleaux formations noted on peripheral blood smear
Renal insufficiency with associated serum protein elevation
Unexplained pathologic fracture or lytic lesion identified on radiograph
Bence Jones proteinuria
NORMAL PATTERN OF SERUM
ABNORMAL PATTERN OF SERUM
MULTIPLE MYELOMAMULTIPLE MYELOMA
Extra M-Band is seen Albumin
Β-globulin and α2-gloublin Albumin
Absence or decrease of γ-globulin and others normal
γ β -globulin
Characteristic Patterns of Acute-Reaction Proteins Found on Serum Protein
Electrophoresis and Associated Conditions or Disorders
Chronic cachectic or wasting diseases
Hemorrhage, burns, or protein-losing enteropathies
Impaired liver function resulting from decreased synthesis of albumin
Increased alpha1 globulins
Decreased alpha1 globulins
Increased alpha2 globulins
Advanced diabetes mellitus
Decreased alpha2 globulins
Severe liver disease
Differential Diagnosis of Polyclonal Gammopathy
Viral infections, especially hepatitis, human
immunodeficiency virus infection,
mononucleosis, and varicella
Focal or systemic bacterial infections,
including endocarditis, osteomyelitis, and
Connective tissue diseases
Systemic lupus erythematosus
Mixed connective tissue
Primary biliary cirrhosis
Primary sclerosing cholangitis
Hematologic cancers (see below)
Hematologic and lymphoproliferative disorders
Sickle cell anemia
Other inflammatory conditions
Gastrointestinal conditions, including ulcerative
colitis and Crohn's disease
Pulmonary disorders, including bronchiectasis,
cystic fibrosis, chronic bronchitis, and
Endocrine diseases, including Graves' disease
and Hashimoto's thyroiditis
Characteristic Features of Monoclonal Gammopathies
Disease Distinctive features
Multiple myeloma M protein appears as a narrow spike in the gamma, beta, or alpha2 regions.
M-protein level is usually greater than 3 g per dL.
Skeletal lesions (e.g., lytic lesions, diffuse osteopenia, vertebral compression fractures)
are present in 80 percent of patients.
Diagnosis requires 10 to 15 percent plasma cell involvement on bone marrow biopsy.
Anemia, pancytopenia, hypercalcemia, and renal disease may be present.
Monoclonal gammopathy of
M-protein level is less than 3 g per dL.
There is less than 10 percent plasma cell involvement on bone marrow biopsy.
Affected patients have no M protein in their urine, no lytic bone lesions, no anemia, no
hypercalcemia, and no renal disease.
Smoldering multiple myeloma M-protein level is greater than 3 g per dL.
There is greater than 10 percent plasma cell involvement on bone marrow biopsy.
Affected patients have no lytic bone lesions, no anemia, no hypercalcemia, and no renal
Plasma cell leukemia Peripheral blood contains more than 20 percent plasma cells.
M-protein levels are low.
Affected patients have few bone lesions and few hematologic disturbances.
This monoclonal gammopathy occurs in younger patients.
Solitary plasmacytoma Affected patients have only one tumor, with no other bone lesions and no urine or serum
WaldenstrÃ¶m's macroglobulinemia IgM M protein is present.
Affected patients have hyperviscosity and hypercellular bone marrow with extensive
infiltration by lymphoplasma cells.
Heavy chain disease The M protein has an incomplete heavy chain and no light chain.
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