M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

Introduction

SERUM PROTEINSCompositionAlbumin: 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 AADecresed Alb cirrhosis,nephrotic syndrome, malnutrition.6. Increased alb - dehydration

GlobulinsIts a glycoprotein with m.w. 90000 130000.Types.The & 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%) 1-3% 2-11% -11% -15%A:G Ratio:1.5:1.

METHODS FOR SEPARATION OF SERUM PROTEINSPrecipitation by salts.Cohns fractional precipitation method.Sedimentation by ultracentrifugation.Paper chromatography.Electrophoresis.

ELECTROPHORESISDefinition: 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 mediuma. Agar gel electrophoresis (AGE).b. PAGE, SDS PAGE, QPNC PAGE (Quantitative preparative native continuous PAGE).c. Cellulose acetate electrophoresis.d. Capillary electrophoresis.

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.

FACTORS AFFECTING ELECTROPHORESISThe electric field:Voltage-V MCurrent-C MResistance-R 1/ M

The sample:Charge-C MSize-S 1/ MShape-Molecules of similar size but different shape such as fibrusand 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.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.

Types of buffers used in electrophoresisTris buffer.Glycine buffer.Sodium barbituric acid.TAE buffer (Tris acidic acid EDTA).

Types of StainsFor serum proteins Amido block- Coomassie brilliant blueFor isoenzymes- Nitro tetra zolium blueFor lipoprotein zones- Fat red 7B- Oil red O- Sudan block BFor DNA fragments- Ethidium bromideFor 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 moleculesSome gels are made with acrylamide if sharper bands are required

Buffer - in this case TBEThe 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 chamberGel chambers come in a variety of models, from commercial through home-made, and a variety of sizes

A gel being runAgarose blockPositive electrodeDNA loaded inwells in the agaroseBlack backgroundTo make loading wells easierCombBuffer

The comb is removed, leaving little wells and buffer is poured over the gel to cover it completelyThe 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 1Indications for Serum Protein Electrophoresis

Suspected multiple myeloma, Waldenstrm's macroglobulinemia, primary amyloidosis, or related disorderUnexplained peripheral neuropathy (not attributed to longstanding diabetes mellitus, toxin exposure, chemotherapy, etc.)New-onset anemia associated with renal failure or insufficiency and bone painBack pain in which multiple myeloma is suspectedHypercalcemia attributed to possible malignancy (e.g., associated weight loss, fatigue, bone pain, abnormal bleeding)Rouleaux formations noted on peripheral blood smearRenal insufficiency with associated serum protein elevationUnexplained pathologic fracture or lytic lesion identified on radiographBence Jones proteinuria

NORMAL PATTERN OF SERUM ELECTROPHORESIS1

i-globulinorigin2-globulin1-globulin2-globulin1-globulinalbumin

ABNORMAL PATTERN OF SERUM ELECTROPHORESISMULTIPLE MYELOMA

iExtra M-Band is seen Albumin

NEPHROTIC SYNDROME

i-globulin and 2-gloublin Albumin

AGAMMAGLOBULINEMIA

iAbsence or decrease of -globulin and others normal

LIVER DISEASES

i -globulinorigin2-globulin1-globulinalbumin

Characteristic Patterns of Acute-Reaction Proteins Found on Serum Protein Electrophoresis and Associated Conditions or Disorders Increased albuminDehydrationDecreased albuminChronic cachectic or wasting diseasesChronic infectionsHemorrhage, burns, or protein-losing enteropathiesImpaired liver function resulting from decreased synthesis of albuminMalnutritionNephrotic syndromePregnancyIncreased alpha1 globulinsPregnancyDecreased alpha1 globulinsAlpha1-antitrypsin deficiencyIncreased alpha2 globulinsAdrenal insufficiencyAdrenocorticosteroid therapyAdvanced diabetes mellitusNephrotic syndromeDecreased alpha2 globulinsMalnutritionMegaloblastic anemiaProtein-losing enteropathiesSevere liver diseaseWilson's disease

Increased beta1 or beta2 globulinsBiliary cirrhosisCarcinoma (sometimes)Cushing's diseaseDiabetes mellitus (some cases)HypothyroidismIron deficiency anemiaMalignant hypertensionNephrosisPolyarteritis nodosaObstructive jaundiceThird-trimester pregnancyDecreased beta1 or beta2 globulinsProtein malnutritionIncreased gamma globulinsAmyloidosisChronic infections (granulomatous diseases)Chronic lymphocytic leukemiaCirrhosisHodgkin's diseaseMalignant lymphomaMultiple myelomaRheumatoid and collagen diseases (connective tissue disorders)Waldenstrm's macroglobulinemiaDecreased gamma globulinsAgammaglobulinemiaHypogammaglobulinemia

Differential Diagnosis of Polyclonal Gammopathy InfectionsViral infections, especially hepatitis, human immunodeficiency virus infection, mononucleosis, and varicellaFocal or systemic bacterial infections, including endocarditis, osteomyelitis, and bacteremiaTuberculosisConnective tissue diseasesSystemic lupus erythematosusMixed connective tissueTemporal arteritisRheumatoid arthritisSarcoidLiver diseasesCirrhosisEthanol abuseAutoimmune hepatitisViral-induced hepatitisPrimary biliary cirrhosisPrimary sclerosing cholangitis MalignanciesSolid tumorsOvarian tumorsLung cancerHepatocellular cancerRenal tumorsGastric tumorsHematologic cancers (see below)Hematologic and lymphoproliferative disordersLymphomaLeukemiaThalassemiaSickle cell anemiaOther inflammatory conditionsGastrointestinal conditions, including ulcerative colitis and Crohn's diseasePulmonary disorders, including bronchiectasis, cystic fibrosis, chronic bronchitis, and pneumonitisEndocrine diseases, including Graves' disease and Hashimoto's thyroiditis

Characteristic Features of Monoclonal Gammopathies

DiseaseDistinctive featuresMultiple myelomaM 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 significanceM-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 myelomaM-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 leukemiaPeripheral 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 plasmacytomaAffected patients have only one tumor, with no other bone lesions and no urine or serum abnormalities.Waldenstrm's macroglobulinemiaIgM M protein is present.Affected patients have hyperviscosity and hypercellular bone marrow with extensive infiltration by lymphoplasma cells.Heavy chain diseaseThe M protein has an incomplete heavy chain and no light chain.