Electro Phor

8/10/2019 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".

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