SERUM PROTEIN ELECTROPHORESIS & THEIR CLINICAL IMPORTANCE

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ELECTROPHORESIS SHOWS SERUM PROTEINS

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

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Introduction
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SERUM PROTEINS

• Composition
• Albumin Conc. 60, M.W. 69000, 585 AAs with 17
  disulphide bonds.
• Synthesized from liver.
• Maintains colloidal osmotic pressure.
• Decreasing causes edema.
• Serves as asource of AA
• Decresed Alb cirrhosis,nephrotic syndrome,
  malnutrition.
• 6. Increased alb - dehydration

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• Globulins
• Its a glycoprotein with m.w. 90000 130000.
• Types.
• The a ß helps to transport proteins, hormones,
  vitamins, minerals and lipids.
• ? 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)
• a1 - 3
• a2 - 11
• ß - 11
• ? - 15
• AG Ratio 1.51.

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METHODS FOR SEPARATION OF SERUM PROTEINS

1. Precipitation by salts.
2. Cohns fractional precipitation method.
3. Sedimentation by ultracentrifugation.
4. Paper chromatography.
5. Electrophoresis.

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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
• 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.

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• 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
• Separating serum proteins for diagnostic purpose.
• Haemoglobin separation.
• Lipoprotein separation and identification.
• Isoenzyme separation and their analysis.
• Nucleic acid studies.
• Determination of molecular weight of the proteins.

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FACTORS AFFECTING ELECTROPHORESIS

• The electric field
• Voltage - V 8 M
• Current - C 8 M
• Resistance- R 1/8 M
• The sample
• Charge - C 8 M
• Size - S 1/8 M
• 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.

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• 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.
• 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.
• 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.
• pH For organic compounds pH determines the
  extent of ionization and therefore degree and
  direction of migration are pH dependent.
• 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.

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Types of buffers used in electrophoresis

1. Tris buffer.
2. Glycine buffer.
3. Sodium barbituric acid.
4. TAE buffer (Tris acidic acid EDTA).

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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

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Gel Electrophoresis serum proteins
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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

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• 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

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• 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

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A gel being run
Positive electrode
Comb
Agarose block
DNA loaded in wells in the agarose
Buffer
Black background To make loading wells easier
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• 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

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• 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

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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

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Pulsed field gel electrophoresis
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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.

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Gel Electrophoresis
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CAPILLARY ELECTROPHORESIS
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MODES OF CAPILLARY ELECTROPHORESIS
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TABLE 1
Indications for Serum Protein Electrophoresis




Suspected multiple myeloma, WaldenstrÃm'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
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NORMAL PATTERN OF SERUM ELECTROPHORESIS

• 1

origin
ß2-globulin
a2-globulin
albumin
i



ß1-globulin
a1-globulin
?-globulin
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ABNORMAL PATTERN OF SERUM ELECTROPHORESIS

• MULTIPLE MYELOMA

i



Extra M-Band is seen Albumin
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NEPHROTIC SYNDROME
i



?-globulin and a2-gloublin Albumin
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AGAMMAGLOBULINEMIA
i



Absence or decrease of ?-globulin and others
normal
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• LIVER DISEASES

origin
a2-globulin
albumin
i


a1-globulin
? ß -globulin
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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 M
egaloblastic anemia Protein-losing
enteropathies Severe liver disease Wilson's
disease
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Increased beta1 or beta2 globulins Biliary
cirrhosis Carcinoma (sometimes) Cushing's
disease Diabetes mellitus (some
cases) Hypothyroidism Iron deficiency
anemia Malignant hypertension Nephrosis Polyarteri
tis 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) WaldenstrÃ
m's macroglobulinemia Decreased gamma
globulins Agammaglobulinemia Hypogammaglobulinemia
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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 Gastroin
testinal 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  
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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.
WaldenstrÃm'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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