Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 76 Methods Of Protein Separation Characterization And Its Clinical Significance Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
Methods of Protein separation, characterization
and its clinical significance
Specific learning objectives
Electrophoresis: Agarose and Polyacrylamide
? Electrophoresis Pattern for Plasma Proteins
? Normal and Monoclonal Gammapathy Pattern
Polyacrylamide Gel Electrophoresis (PAGE) divided into
Native PAGE and SDS-PAGE
? Immunoblotting
Isoelectric Focusing
Two-Dimensional Electrophoresis
Electrophoresis
Technique for separating charged molecules such as aa, proteins,
nucleic acids in a mixture under influence of applied electric field
Charged molecules in electric field move at a speed determined by
their charge to mass ratio
Two types of electrophoresis: Moving boundary (electrophoresis in
free solution) used for analysis but not for fractionation of complex
mixture but
Zone (sample is constrained to move in solid support i.e. filter paper
or a gel called gel electrophoresis
Agarose gel electrophoresis
Agarose is natural colloid extracted from seaweed, it's a linear
polysaccharide made up of basic repeat unit agarobiose, which
consists of alternate units of galactose and 3,6-anhydrogalactose
Large pore size and used to separate very large molecules
(>200kDa)
Used for electrophoresis of both proteins and nucleic acid
Electrophoresis Pattern for Plasma Proteins
Major peaks observed based on their migration are those of albumin,
, , and -globulins, fibrinogen, and and globulins.
1
2
1
2
Some of these peaks represents tens to hundreds of different plasma
proteins that have a similar migration rate at pH 8.6.
Certain proteins predominate in each peak and variation in their
relative amounts is characteristic of certain diseases.
Fig. 2.20. Textbook of Biochemistry with Clinical Correlations, 4th Ed
by Thomas M Devlin
? Monoclonal gammapathies are due
to clonal synthesis of a unique Ig
and give rise to a sharp -globulin
band pattern
Fig. 2.21. Textbook of Biochemistry with Clinical Correlations, 4th edition by Thomas M Devlin
Serum Protein Electrophoresis (SPEP)
Serum is applied on a support medium and exposed to an electric
current
Different fractions of serum proteins separate usually into 5 bands, as-
albumin, 1, 2 , , and -globulin fractions.
Interpretation of SPEP to region, because it mainly composed of Ig.
Cont--
Increase in region , shows homogenous spike like a peak in -
globulin zone, in case of monoclonal gammapathies (MG).
Result from proliferation of a single, malignant clone of plasma cells
which produce either a single class of intact Igs, heavy and light chains
or both.
These proteins are called M (monoclonal) proteins, detected as a sharp
symmetric spike (M spike) with an 2, , or a mobility.
Cont--
Normally, plasma cells constitute 1% of cells in bone marrow, but as
disease progress, tumor load in bone marrow increases up to 80%,
depends upon disease severity.
Malignant plasma cells synthesize monoclonal antibodies which are
released into circulation and its level increases in serum.
Normal and Monoclonal Gammapathy Pattern
Fig.2 & 3: Tripathy S et al 2012
Table.18.6.Clinical Biochemistry, by Nessar Ahmed
Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel consists of chains of acrylamide monomers cross-
linked with N,N'-methylene-bisacrylamide units called bis
Pore size of gel determined by both total conc of monomers
(acrylamide + bis) and ratio of acrylamide to bis
Polymerization of acrylamide: bis solution initiated by APS
(ammonium persulfate) and catalyzed by TEMED (N,N,N',N'-
tetramethylethylenediamine)
Cont--
It has high resolving power for small and moderately sized proteins
and nucleic acids (upto 1x106 Da)
Migration of a protein in a gel during electrophoresis based on a
charge density, size or mass and its shape
If two proteins have same size or mass and shape, one with greatest
charge density move faster through gel, similarly, if two proteins
having same charge density and shape, one with smaller size or
mass migrate faster than large size protein
Fig 3.19:Harper's Illustrated Biochemistry, 30th Ed.
SDS-PAGE
Proteins exposed to negative charged anionic detergent SDS before
and during gel electrophoresis
SDS binds to main chains at ratio of one SDS for every two aa, which
imparts large net negative charge on protein.
Negative charge acquired by protein due to binding of SDS is much
greater than charge on native protein, this native charge thus
becomes insignificant
Cont--
If protein itself has very large positive or negative charge, this charge
may not be negligible compared with charge produced by bound SDS
Protein treat with SDS have similar charge to mass ratio due to
amount of SDS bound per unit weight of protein is constant 1.4 g of
SDS/gm of protein
Cont--
SDS treatment eliminates effect of differences in shape and charge
density so that chain length, reflects mass is sole determinant of
migration rate of proteins in SDS-PAGE
Separating gel used is 15% polyacrylamide gel for separating
proteins in range of 10-100kDa, if molecular mass >100kDa then
large pore sized gel (10% polyacrylamide gel) would be used
How to estimate molecular weight of a protein?
Standard proteins of known molecular weight (Proteins marker) used to
estimate molecular weight (Mwt.) of an unknown protein.
Position of an unidentified protein provide an measure of its Mwt.
compared with positions to which standard proteins of known Mwt.
migrate in gel.
If protein has two or more different subunits, subunits separated by SDS
treatment and a separate band wil appear for each.
SDS-PAGE
Fig 3.20: Lehninger Principles of Biochemistry by David L Nelson
Immunoblotting
To determine the size and amount of the protein in given sample
Diagnosis of diseases, to detect antibody against virus or bacteria in
serum
Confirmatory test for HIV, detects anti-HIV antibody in patient's serum
Detect defective proteins i.e. prion disease
Fig.6.23. Immunoblotting procedure: Biochemistry. 4th edition by Donald Voet and Judith G. Voet
Isoelectric Focusing (IF)
IF: If a mixture of proteins is electrophoresed through a solution having a
stable pH gradient in which pH smoothly increases from anode to cathode,
each protein wil migrate to position in pH gradient corresponding to its
isoelectric point
Used to determine isoelectric point (pI) of a protein
pH gradient obtained by allow a mixture of low Mwt. organic acids and
bases to distribute themselves in an electric field generated across gel.
Fig 3.21: Lehninger Principles of Biochemistry by David L Nelson
Two-Dimensional Electrophoresis
Sequential combination of isoelectric focusing and SDS
electrophoresis in a process called two-dimensional (2-D)
electrophoresis permits resolution of complex mixtures of proteins.
2-D electrophoresis separates proteins of identical Mwt. that differ in
pI, or proteins with similar pI values but different Mwt.
Fig 3.22 (a): Lehninger Principles of Biochemistry by David L Nelson
Fig 3.22 (b): Lehninger Principles of Biochemistry by David L Nelson
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This post was last modified on 05 April 2022