Electro Phor
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Transcript of Electro Phor
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ELECTROPHORETIC METHODS
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1. It is the process of moving charged
biomolecules in solution by applyingan electrical field across the mixture.
2. Biomolecules moved with a speed
dependent on their charge, shape,
and size and separation occures onthe basis of molecular size.
Electrophoresis is used for analysis and
purification of very large molecules
!proteins, nucleic acids"
for analysis of simpler charged
molecules !sugars, amino acids,
peptides, nucleotides, and simpler
ions".
Basic principles of electrophoresis
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#hen charged molecules are
placed in an electric field,they migrate toward eitherthe positive !anode" ornegative !cathode" poleaccording to their charge.
1. $actors influencedelectrophoresis mobility
2. net charge of the molecule
%. size and shape
&. concentration of themolecule in solution
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Amino acids have characteristic titration curves
Fully protonated
form at wery low pH
At the midpoint pK1=2.34 there
is equimolar concentration of
proton donor and proton acceptor.
Dipolar ion
At the midpoint pK=!."# there
is equimolar concentration of
proton donor and protonacceptor.
$roton
donor
$roton
acceptor
$roton
donor
$roton
acceptor
Izoelectric point
Adopted from:D.L. Nelson, M.M. CoxLehninger Principle of Biochemistr
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Electrophoresis is carried out by applying a thin
layer
'(ueous protein solution is immobilized in a solid hydrophilic
support.
)olid matrix with pores which are used
* paper* starch* cellulose acetate* polyacrylamide
* agar+agarose
olecules in the sample move through porous matrix at
different velocity.
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* Electrophoresis can be one
dimensional !i.e. one plane of
separation" or two dimensional.
* -ne dimensional electrophoresis is
used for most routine protein and
nucleic acid separations. wodimensional separation of proteins is
used for finger printing , and when
properly constructed can be
extremely accurate in resolving all of
the proteins present within a cell!greater than 1,/00".
* ost common stabilizing media are
polyacrylamide or agarose gels.
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* $unction of buffer1. carries the applied current
2. established the p
%. determine the electric charge on the solute
* igh ionic strength of buffer produce sharper band
produce more heat
* 3ommonly used buffer* Barbital buffer 4 ris5E6' for protein
* ris5acetate5E6' 4 ris5borate5E6' !/0mmol+78 p 9./59.:"
Buffers
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Zone electrophoresis
* uch simple method* uch greater resolution* ;e(uire small sample* 'cetate cellulose support medium
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)tripe of cellulose acetate
Electrophoresis
a>or protein components
separate into discrete zones
6ensitometer tracing density of zones is proportional
to the amount of protein
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Example of application of zone electrophoresis in
clinical practice
ypergamaglobulinemia
ypogamaglobulinemia
=ormal serum
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* ?el is a colloid in a solid form !@@A is water".
* ?el material acts as a molecular sieve.
* 6uring electrophoresis, macromolecules are forced to
move through the pores when the electrical current is
applied.
Gel electrophoresis
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* 'garose and polyacrylamide gels are across5linCed, spongeliCe
structure
* It is important that the support media is electrically neutral.
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Agarose highly purified
polysaccharide derived from
agar !extracted from
seeweed", long sugar
polymers held together byhydrogen and hydrophobic
bonds.
Acrylamide!32F353-5=2"
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* $or the separation of !1" large protein or protein
complex !2" polynucleotide /05%0,000 base5pairs
* he pore size is determined by ad>usting the
concentration of agarose in a gel !normally in the ranCof 0.&5&A
Agarose gels
OH
OOH
CH2OHO
O OH
O
O
O
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CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO
NH
CH2NH
CO
CO
NH
CH2
NH
CO
CO
NH2
NH2
CO
n
n
Polyacrylamide gels
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SDS-polyacrylamide gel electrophoresis (SDS-PAGE"
* )6) !also called lauryl sulfate" 5 anionic detergent
* olecules in solution with )6) have a net negative charge within a wide prange.
* ' polypeptide chain binds amounts of )6) in proportion to its relative
molecular mass.* he negative charges on )6) destroy most of the complex structure of
proteins, and are strongly attracted toward an anode !positively5charged
electrode" in an electric field.
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6iagrams of vertical slab gel assembly
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6etermination of molecular mass
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)tain 6etection limit
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)taining with 3oomasie blue
! " #
!
"
#
'ssesment of individual lines
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'n ethidium5stained gel photographed under GH light
Each band that you see is a collection of millions of
6=' molecules, all of the same lengthJJ
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estern blott techni!ue
* #estern blot !also called immunoblot"
is a techni(ue to detect specifically one
protein in a mixture of large number of
proteins and to obtain information about
the size and relative amounts of the
protein present in different samples.
* In first proteins are separated using
)6)5polyacrylamide gel electrophoresis"
* hen they are moved onto a
nitrocellulose membrane. he proteins
retain the same pattern of separation
they had on the gel.
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* 'n antibody is then added to the
solution which is able to bind to its specific
protein and forms an antibody5protein
complex with the protein of interest. !In
fact there is no room on the membrane for
the antibody to attach other than on the
binding sites of the specific target protein".
* $inally the nitrocellulose membrane is
incubated with a secondary antibody,
which is an antibody5enzyme con>ugate
that is directed against the primary
antibody.
* he location of the antibody is revealed
by incubating it with a substrate that the
attached enzyme converts to a product
that can be seen and followed and then
photographed.
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#soelectric focusation
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$%o-dimensional gel electrophoresis
!&-D electrophoresis"
In the first dimension, proteins are resolved in according to their isoelectric
points !pIs" using immobilized p gradient electrophoresis !I
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Electrophoreogram of the mixture of proteins
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Capillary electrophoresis
3apillaries are typically of /0 Mm inner diameter and 0./ to 1 m in
length.6ue to electroosmotic flow, all sample components migrate towards
the negative electrode.
he capillary can also be filled with a gel, which eliminates the
electroosmotic flow. )eparation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution, greatersensitivity, and on5line detection.
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Electroosmotic flo%
he surface of the silicate glass capillary contains negatively5chargedfunctional groups that attract positively5charged counterions. he
positively5charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. his overall solvent
movement is called electroosmotic flow. 6uring a separation,
uncharged molecules move at the same velocity as the electroosmotic
flow !with very little separation".