ELECTROPHORESIS

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ELECTROPHORESIS
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Definition Migration of charged solutes
or particles in a liquid medium under the
influence of an applied electric field. Cations
vely charged Cathode -ve
electrode Anions -vely charged Anode
ve electrode Principle Chemical species
carrying an electrical charge by virtue of
ionization move either to the cathode or anode in
an electrophoresis system, depending on the kind
of the charge they carry.
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pI pH at which the net charge on the molecule is
zero. If pHgt pI -vely charged molecules If
pHltpI vely charged molecules. Electrophoretic
mobility Rate of migration (?). F
Eq/d F- Force operating on the molecule with
charge q E/d field strength applied
E - potential difference d
distance between electrodes.
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Frictional force (F) 6r V r radius of
the molecule viscosity of the medium, V
velocity Eq/d 6r V V Eq/ 6r
V
V Eq
6r
V is proportional to field strength, charge q V
is inversely proportional to particle size(r) and
viscosity of the solution.( )
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Factors affecting the rate of migration

• Net electrical charge on the molecule
• Size and shape of the molecule(Charge/mass ratio)
• Electrical field strength
• Properties of supporting medium
• Temperature of operation
• Viscosity of the solution.

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Apparatus
a. Electrophoretic cell consists of electrodes,

buffer reservoir
Support medium
insulating cover b. Power pack -
provides a stabilized current.
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-

support
electrodes
Power supply
Buffer reservoir
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• Electrophoreto gram
• A display of separated
  components, each sharpely separated from
  neighbouring zone on support materials.
• Buffer
• Solution of a weak acid and its salt
• maintains a constant pH.
• commonly used buffer
• Barbitone
• phosphate buffer
• Tris borate buffer
• Tris acetate EDTA.

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• Buffer serve the following purposes
• Carry the applied current
• Fix the pH
• Determines the charge on the molecule
• Determines to which electrode the solute will
• migrate

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Stains Protein stain - coomassie brilliant
blue - silver nitrate
- Ponceau S Isoenzymes
Nitro tetrazolium blue Lipoproteins Sudan
black - oil red o
- fat red 7.3 DNA -
Ethidium bromide CSF protein Silver nitrate

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TYPES
BASED ON THE SUPPORT MEDIA
Moving boundary electrophoresis
Zone electrophoresis
GEL Polyacrylamide Agarose SDS-PAGE Starch
PAPER Cellulose Cellulose acetate
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General procedure
Materials provided Serum(unhaemolysed) Supporting
media Electrophoresis apparatus Power pack Stain

• Separation
• Staining
• Detection
• quantification

Uses mainly for separating S.protein,
Lipoproteins, Hb, Isoenzymes
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• STEP1
• Separation
• Saturation of cellulose acetate paper with buffer
• Sample tracking dye as a narrow streak.
• Buffer pH 8.6 Tris EDTA Borate buffer
• Current is switched on. 100v 1m Amp
• Proteins are negatively charged. So migrate
  towards anode.
• After the tracking dye has moved to anode side
  the current is switched off.
• 2.Fixing and staining Ponceau S dye in
  trichloracetic acid.
• Destaining the background 5 acetic acid

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• Quanitfication
• Zone containing each band is cut from the paper
  and placed in a suitable solvent.
• Measure the colour intensity in colorimeter.
• B. Densitometer paper is made transparent and
  subjected to densitometry.
• Advantages of cellulose acetate electrophoresis
• No adsorption of sample on support
• Better resolution in a short time.

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Separation pattern Albumin ?1 globulin- ?1
antitrypsin(?1 AT) ?1 acid
glycoprotein ?1
Fetoprotein(AFP) ?2 globulin - Haptoglobin
?2 Macroglobulin
ceruloplasmin ?1 globulin -
Transferrin, C4 ?2 globulin - c3, ?2
microglobulin ? Globulin - IgA, IgM, IgG, CRP
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Acute phase reaction(APR) A non specific
response to inflammation (infection, autoimmune
disease or tissue damage(trauma, surgery, MI,
tumors) Positive acute phase proteins
?1 antitrypsin(?1 AT) ?1 acid glycoprotein
Haptoglobin ceruloplasmin C4,C3,CRP
Negative acute phase reactants Albumin

Transferrin
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Normal
CRF
?Alb
Alb


?1
?2
?2
?1
?2
?
-
-
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IgG monoclonal gammapathy
Normal
2.2g/dl
Alb


?1
?2
?1
?2
?
-
-
Multiple myeloma T. Protein 9gm/dl
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Nephrotic syndrome
Normal
0.6g/dl
Alb


?1
?2
??2
?1
?1
?2
?2
?
?
-
-
T. Protein 2.9g/dl
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Normal
Inflammation
Alb
Alb


?1
??1
?2
??2
?1
?1
?2
?2
?
?
-
-
TP- 5.8g/dl Alb-2.5g/dl
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Chronic hepatic disease
Normal
2.2g/dl
Alb


?1
?2
?1
?1
?2
?2
?
?
-
-
?-? fusion
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Lipoprotein electrophoresis
?chylomicrons
TYPE 1 LPL deficiency
-

LDL
VLDL
HDL
CM
?LDL
TYPE IIA

-
LDL
VLDL
HDL
CM
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?LDL VLDL
TYPE III Broad ?
-

LDL
VLDL
HDL
CM
?VLDL
TYPE IV

LDL
VLDL
HDL
CM
-
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?VLDL CM
TYPE V

LDL
VLDL
HDL
CM
-
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Haemoglobin electrophoresis
Normal
AS
SS
HBA
HBF
HBA2

-
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Polyacrylamide gel electrophoresis

• Cross linked polyacrylamide are formed from the
  polymerization of
• Acrylamide monomer
• Bis acrylamide
• Catalysts ammonium persulphate TEMED.

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Discontinuous gel electrophoresis
Two gel layers Lower
resolving gel Upper
stacking gel
-
Stacking gel 2-3 acrylamide pH 6.9
Resolving gel 7.5 acrylamide pH 8.9

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Upper buffer tank
sample
Lower buffer tank
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-
-
Glycine
Protein
Cl-
Resolved protein


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1. Load the samples dissolved in glycine Cl
buffer pH 8.9. 2. In stacking gel glycine exists
as zwitter ion. chloridegtprotein or nucleic
acidgtglycinate 3. Sample forms a concentrated
band 4. In lower gel, proteins encounter ? in pH
and in ?pore size. 5. Movement
Clgtglycinategtprotein. Small moleculesgtlarge
molecules. Molecular sieving effect. Use
analysing protein mixture
qualitatively checking purity
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SDS-PAGE
If protein molecules have uniform charge,then
mobility is directly proportional on size. Use
molecular weights can be determined. Protein
subunits can be dissociated. Compounds used are
solubilizers Urea, ?mercaptoethanol, sodium
dodecyl sulfate. ?mercaptoethanol- breaks S-S
bonds in oligomeric protein(eg insulin)
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SDS- Disrupts secondary, tertiary and quarternary
structures denatures the protein. Polypeptide
chains are coated with negatively charged SDS
molecules. Larger molecules are retarded by
molecular sieving effect. Smaller molecules
have greater mobility.
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Log mol. wt
mobility
Advantages Thermostable, transparent. Pore size
can be controlled. Demerit potential
carcinogenicity
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Starch gel electrophoresis

• Separation of macromolecular ions based on
  surface charge and molecular size.
• The pore size cannot be controlled
• Contamination with microorganisms
• Opaque- hence densitometer scanning not possible

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Agarose gel electrophoresis
1. Linear polysaccharide with repeating unit of
agarobiose. 2.Prepare 1 agarose solution in
buffer. 3. Boil it and pour it on to a glass
slide. Let it cool. 4. Apply sample with a help
of a cover slip(sample tracking dye) on the
cathode side. 5. Place the slide in
electrophoretic tank prefilled with buffer. 6.
Place wicks to ensure buffer flow.
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7. Switch on the current. 100v 1mamp(1hr). 8.After
the run keep in fixative solution. 9. Dry in an
oven 10. Stain with amido black 11. Removal back
ground staining with acetic acid. Use S.
proteins, Hb variants, LDH CK isoenzymes,
lipoprotein fractions Immunoelectrophoresis Isoele
ctric focussing. Advantage low affinity for
proteins native clarity after
drying densitometry pore
size can be controlled.
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Iso electric focussing
This examines the electrophoretic mobility as a
function of pH. Ideal for separating amphoteric
substance such as protein. Protein below their
pI Positively charged Above pI
- negatively charged At pI
- zero mobility.
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Method of separating proteins according to their
pI in a pH gradient. Single electrophoretic run
in a medium of gradually changing pH. pH gradint
is established by ampholytes

-
H
P
OH-
P
1 2 3 4 5 6 7 8 9
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Direction of migration
Anions Zwitter ions
cations
anion
-

X-
X
X
cations
Immobilized Sample Components at pI
Sample introduced at a pH gtpI
Sample A pHltpI
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Uses 1Widely used for separation of s.
proteins 2.Food agriculture industries forensic
and human genetic labs for research in
enzymology, immunology 3. Studying
microheterogeneity in a protein. SDS
gel 1 band IEF - 2-3
bands 4. Separating isoenzymes
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Two dimensional polyacrylamide gel electrophoresis

• First dimension is IEF separation according to
  charge.(pI)
• Separation according to the size in SDS-PAGE
  2nd dimension.
• Use Resolution of 1000-3000 proteins from a
  cell /tissue extract.
• Highly sophisticated analytical method for
  analyzing protein mixtures.

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Immuno electrophoresis
Based on electrophoretic mobility and precipitin
reaction. (double diffusion) In precipitin
reaction, an antigen combines with its specific
antibody to form antigen-antibody complex. Since
most of the Ag-Ab complexes are insoluble, it can
be visualized with naked eye. IEP exploits the
specificity of the reaction between antigen and
antibody and molecular sieving of the gel in
which the reaction takes place.
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Contro well
-

Direction of the diffusion
Precipitin arc
Patient serum well
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a.Separate the protein by routine protein
electrophoresis on agarose gel. b. Antihuman
serum is placed in the trough. c. The antiserum
and the separated patient proteins diffuse toward
each other. d. Precipitin arcs form at the zone
of equivalence between the antigen and specific
antisera. e. Anti total human serum is a
mixture of antibodies against all serum proteins
and produces many precipitin arcs.
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Uses To detect free light chains To
screen for abnormalities in immunoglobulin
classes Semiquantitative, because the size of arc
indicates amount of immunoglobulin. Ie to
determine the concentration of any antigen. To
investigate the purity or to detect particular
antigen.
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Blotting Techniques
Southern blotting DNA separation Northern
blotting RNA separation Western blotting
Protein separation
Separate fragments by Agarose electrophoresis Tr
ansfer onto a nitrocellulose membrane Detect
with radiolabelled probe( antibody in western)