Electrophoresis: Agarose and Polyacrylamide
? Electrophoresis Pattern for Plasma Proteins
--- Content provided by FirstRanker.com ---
? Normal and Monoclonal Gammapathy PatternPolyacrylamide Gel Electrophoresis (PAGE) divided into
Native PAGE and SDS-PAGE
--- Content provided by FirstRanker.com ---
? Immunoblotting
Isoelectric Focusing
--- Content provided by FirstRanker.com ---
Two-Dimensional ElectrophoresisElectrophoresis
Technique for separating charged molecules such as aa, proteins,
--- Content provided by FirstRanker.com ---
nucleic acids in a mixture under influence of applied electric fieldCharged molecules in electric field move at a speed determined by
their charge to mass ratio
--- Content provided by FirstRanker.com ---
Two types of electrophoresis: Moving boundary (electrophoresis in
free solution) used for analysis but not for fractionation of complex
--- Content provided by FirstRanker.com ---
mixture butZone (sample is constrained to move in solid support i.e. filter paper
or a gel called gel electrophoresis
--- Content provided by FirstRanker.com ---
Agarose gel electrophoresis
Agarose is natural colloid extracted from seaweed, it's a linear
--- Content provided by FirstRanker.com ---
polysaccharide made up of basic repeat unit agarobiose, whichconsists of alternate units of galactose and 3,6-anhydrogalactose
Large pore size and used to separate very large molecules
--- Content provided by FirstRanker.com ---
(>200kDa)
Used for electrophoresis of both proteins and nucleic acid
Electrophoresis Pattern for Plasma Proteins
--- Content provided by FirstRanker.com ---
Major peaks observed based on their migration are those of albumin,
, , and -globulins, fibrinogen, and and globulins.
--- Content provided by FirstRanker.com ---
12
1
--- Content provided by FirstRanker.com ---
2
Some of these peaks represents tens to hundreds of different plasma
--- Content provided by FirstRanker.com ---
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.
--- Content provided by FirstRanker.com ---
Fig. 2.20. Textbook of Biochemistry with Clinical Correlations, 4th Ed
by Thomas M Devlin
? Monoclonal gammapathies are due
--- Content provided by FirstRanker.com ---
to clonal synthesis of a unique Ig
and give rise to a sharp -globulin
--- Content provided by FirstRanker.com ---
band patternFig. 2.21. Textbook of Biochemistry with Clinical Correlations, 4th edition by Thomas M Devlin
Serum Protein Electrophoresis (SPEP)
--- Content provided by FirstRanker.com ---
Serum is applied on a support medium and exposed to an electric
current
--- Content provided by FirstRanker.com ---
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.
--- Content provided by FirstRanker.com ---
Cont--Increase in region , shows homogenous spike like a peak in -
globulin zone, in case of monoclonal gammapathies (MG).
--- Content provided by FirstRanker.com ---
Result from proliferation of a single, malignant clone of plasma cells
which produce either a single class of intact Igs, heavy and light chains
--- Content provided by FirstRanker.com ---
or both.These proteins are called M (monoclonal) proteins, detected as a sharp
symmetric spike (M spike) with an 2, , or a mobility.
--- Content provided by FirstRanker.com ---
Cont--
Normally, plasma cells constitute 1% of cells in bone marrow, but as
--- Content provided by FirstRanker.com ---
disease progress, tumor load in bone marrow increases up to 80%,depends upon disease severity.
Malignant plasma cells synthesize monoclonal antibodies which are
--- Content provided by FirstRanker.com ---
released into circulation and its level increases in serum.
Normal and Monoclonal Gammapathy Pattern
Fig.2 & 3: Tripathy S et al 2012
--- Content provided by FirstRanker.com ---
Table.18.6.Clinical Biochemistry, by Nessar Ahmed
Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel consists of chains of acrylamide monomers cross-
--- Content provided by FirstRanker.com ---
linked with N,N'-methylene-bisacrylamide units called bis
Pore size of gel determined by both total conc of monomers
--- Content provided by FirstRanker.com ---
(acrylamide + bis) and ratio of acrylamide to bisPolymerization of acrylamide: bis solution initiated by APS
(ammonium persulfate) and catalyzed by TEMED (N,N,N',N'-
--- Content provided by FirstRanker.com ---
tetramethylethylenediamine)
Cont--
--- Content provided by FirstRanker.com ---
It has high resolving power for small and moderately sized proteinsand nucleic acids (upto 1x106 Da)
Migration of a protein in a gel during electrophoresis based on a
--- Content provided by FirstRanker.com ---
charge density, size or mass and its shape
If two proteins have same size or mass and shape, one with greatest
--- Content provided by FirstRanker.com ---
charge density move faster through gel, similarly, if two proteinshaving same charge density and shape, one with smaller size or
mass migrate faster than large size protein
--- Content provided by FirstRanker.com ---
Fig 3.19:Harper's Illustrated Biochemistry, 30th Ed.SDS-PAGE
Proteins exposed to negative charged anionic detergent SDS before
--- Content provided by FirstRanker.com ---
and during gel electrophoresis
SDS binds to main chains at ratio of one SDS for every two aa, which
--- Content provided by FirstRanker.com ---
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
--- Content provided by FirstRanker.com ---
becomes insignificant
Cont--
If protein itself has very large positive or negative charge, this charge
--- Content provided by FirstRanker.com ---
may not be negligible compared with charge produced by bound SDS
Protein treat with SDS have similar charge to mass ratio due to
--- Content provided by FirstRanker.com ---
amount of SDS bound per unit weight of protein is constant 1.4 g ofSDS/gm of protein
Cont--
--- Content provided by FirstRanker.com ---
SDS treatment eliminates effect of differences in shape and charge
density so that chain length, reflects mass is sole determinant of
--- Content provided by FirstRanker.com ---
migration rate of proteins in SDS-PAGESeparating gel used is 15% polyacrylamide gel for separating
proteins in range of 10-100kDa, if molecular mass >100kDa then
--- Content provided by FirstRanker.com ---
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
--- Content provided by FirstRanker.com ---
estimate molecular weight (Mwt.) of an unknown protein.
Position of an unidentified protein provide an measure of its Mwt.
--- Content provided by FirstRanker.com ---
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
--- Content provided by FirstRanker.com ---
treatment and a separate band wil appear for each.
SDS-PAGE
--- Content provided by FirstRanker.com ---
Fig 3.20: Lehninger Principles of Biochemistry by David L NelsonImmunoblotting
To determine the size and amount of the protein in given sample
Diagnosis of diseases, to detect antibody against virus or bacteria in
--- Content provided by FirstRanker.com ---
serum
Confirmatory test for HIV, detects anti-HIV antibody in patient's serum
Detect defective proteins i.e. prion disease
--- Content provided by FirstRanker.com ---
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
--- Content provided by FirstRanker.com ---
stable pH gradient in which pH smoothly increases from anode to cathode,
each protein wil migrate to position in pH gradient corresponding to its
--- Content provided by FirstRanker.com ---
isoelectric pointUsed to determine isoelectric point (pI) of a protein
pH gradient obtained by allow a mixture of low Mwt. organic acids and
--- Content provided by FirstRanker.com ---
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
--- Content provided by FirstRanker.com ---
Sequential combination of isoelectric focusing and SDS
electrophoresis in a process called two-dimensional (2-D)
--- Content provided by FirstRanker.com ---
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.
--- Content provided by FirstRanker.com ---
Fig 3.22 (a): Lehninger Principles of Biochemistry by David L Nelson
Fig 3.22 (b): Lehninger Principles of Biochemistry by David L Nelson
Thank you
--- Content provided by FirstRanker.com ---