M.

PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.


Introduction
SERUM PROTEINS
Composition
Albumin: Conc. 60%, M.W. 69000, 585 AAs with 17 disulphide
bonds.
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
Globulins
1. Its a glycoprotein with m.w. 90000 130000.
2. Types.
3. The & helps to transport proteins, hormones, vitamins, minerals
and lipids.
4. 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%)

1
- 3%

2
- 11%
- 11%
- 15%
A:G Ratio: 1.5:1.



METHODS FOR SEPARATION OF SERUM
PROTEINS
1. Precipitation by salts.
2. Cohns fractional precipitation method.
3. Sedimentation by ultracentrifugation.
4. Paper chromatography.
5. Electrophoresis.
ELECTROPHORESIS
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).

Types:
1. Depending upon the nature of supporting medium
a. Agar gel electrophoresis (AGE).
b. PAGE, SDS PAGE, QPNC PAGE (Quantitative preparative native
continuous PAGE).
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 Stains
For 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
molecules
Some gels are made with
acrylamide if sharper
bands are required
Buffer - in this case
TBE
The buffer provides
ions in solution to
ensure electrical
conductivity.
Not only is the agarose
dissolved in buffer, but
the gel slab is
submerged (submarine
gel) in buffer after
hardening
Also needed are a
power supply and a
gel chamber
Gel chambers come
in a variety of
models, from
commercial through
home-made, and a
variety of sizes
A gel being run
Agarose block
Positive electrode
DNA loaded in
wells in the agarose
Black background
To make loading wells easier
Comb
Buffer
The comb is
removed, leaving
little wells and
buffer is poured over
the gel to cover it
completely
The serum samples
are mixed with a
dense loading dye
so they sink into their
wells and can be
seen
The serum samples
are put in the wells
with a micropipette.
Micropipettes have
disposable tips and
can accurately
measure
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
genetic fingerprinting.
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
Pulsed field gel electrophoresis

SDS-PAGE

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
mobility .
Quantitative preparative native continuous
polyacrylamide gel electrophoresis (QPNC-PAGE) is a
new method for separating native metalloproteins in
complex biological matrices.
Gel Electrophoresis
CAPILLARY ELECTROPHORESIS
MODES OF CAPILLARY
ELECTROPHORESIS
TABLE 1
Indications for Serum Protein Electrophoresis
Suspected multiple myeloma, Waldenstrm'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
ELECTROPHORESIS
1









i
-globulin
origin
2-globulin

1
-globulin

2-globulin

1-globulin

albumin
ABNORMAL PATTERN OF SERUM
ELECTROPHORESIS
MULTIPLE MYELOMA









i
Extra M-Band is seen Albumin

NEPHROTIC SYNDROME









i
-globulin and
2
-gloublin Albumin
AGAMMAGLOBULINEMIA









i
Absence or decrease of -globulin and others normal
LIVER DISEASES






i

-globulin
origin

2-globulin

1-globulin

albumin
Characteristic Patterns of Acute-Reaction Proteins Found on Serum Protein
Electrophoresis and Associated Conditions or Disorders
Increased albumin
Dehydration
Decreased albumin
Chronic cachectic or wasting diseases
Chronic infections
Hemorrhage, burns, or protein-losing enteropathies
Impaired liver function resulting from decreased synthesis of albumin
Malnutrition
Nephrotic syndrome
Pregnancy
Increased alpha1 globulins
Pregnancy
Decreased alpha1 globulins
Alpha1-antitrypsin deficiency
Increased alpha2 globulins
Adrenal insufficiency
Adrenocorticosteroid therapy
Advanced diabetes mellitus
Nephrotic syndrome
Decreased alpha2 globulins
Malnutrition
Megaloblastic anemia
Protein-losing enteropathies
Severe liver disease
Wilson's disease
Increased beta1 or beta2 globulins
Biliary cirrhosis
Carcinoma (sometimes)
Cushing's disease
Diabetes mellitus (some cases)
Hypothyroidism
Iron deficiency anemia
Malignant hypertension
Nephrosis
Polyarteritis nodosa
Obstructive jaundice
Third-trimester pregnancy
Decreased beta1 or beta2 globulins
Protein malnutrition
Increased gamma globulins
Amyloidosis
Chronic infections (granulomatous diseases)
Chronic lymphocytic leukemia
Cirrhosis
Hodgkin's disease
Malignant lymphoma
Multiple myeloma
Rheumatoid and collagen diseases (connective tissue disorders)
Waldenstrm's macroglobulinemia
Decreased gamma globulins
Agammaglobulinemia
Hypogammaglobulinemia
Differential Diagnosis of Polyclonal Gammopathy
Infections
Viral infections, especially hepatitis, human
immunodeficiency virus infection,
mononucleosis, and varicella
Focal or systemic bacterial infections,
including endocarditis, osteomyelitis, and
bacteremia
Tuberculosis
Connective tissue diseases
Systemic lupus erythematosus
Mixed connective tissue
Temporal arteritis
Rheumatoid arthritis
Sarcoid
Liver diseases
Cirrhosis
Ethanol abuse
Autoimmune hepatitis
Viral-induced hepatitis
Primary biliary cirrhosis
Primary sclerosing cholangitis
Malignancies
Solid tumors
Ovarian tumors
Lung cancer
Hepatocellular cancer
Renal tumors
Gastric tumors
Hematologic cancers (see below)
Hematologic and lymphoproliferative disorders
Lymphoma
Leukemia
Thalassemia
Sickle cell anemia
Other inflammatory conditions
Gastrointestinal conditions, including ulcerative
colitis and Crohn's disease
Pulmonary disorders, including bronchiectasis,
cystic fibrosis, chronic bronchitis, and
pneumonitis
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
undetermined significance
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
disease.
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
abnormalities.
Waldenstrm'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.