4. INTRODUCTION
Electrophoresis is the migration of charged particles
or molecules in a medium under the influence of an
applied electric field.
4
5. principle
The rate of migration of an ion in electrical field depend on
factors,
o Net charge of molecule
o Size and shape of particle
o Strength of electrical field
o Properties of supporting medium
o Temperature of operation
o Ionic strength of buffer
6.
7.
8.
9. Effects of heating
Peak broadening of samples
Decrease resolution
Formation of temperature gradient –convection current -
resulting mixing of analytes
Denaturation of enzymes and proteins
Effect of heat
19. Buffer
Thebuffer in electrophoresis hastwofold purpose:
Carry appliedelectrical current
Theyset the pH atwhichelectrophoresis iscarried out.
Thusthey determine;
Typeof charge on solute.
Extent of ionizationof solute
Electrode towards whichthe solute will migrate.
The buffer ionic strength will determine the thickness of the
ionic cloud.
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20.
21.
22. Commonly buffers used;
Buffer
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pH value
Phosphate buffer around 7.0
Tris-Borate-EDTAbuffer (TBE) around 8.0
Tris-Acetate EDTAbuffer(TAE) above 8.0
TrisGlycine buffer (TG) more than 8.5
Tris-Citrate-EDTAbuffer (TCE) around 7.0
Tris-EDTAbuffer (TE) around 8.0
Tris-Maleic acid -EDTAbuffer (TME) around 7.5
Lithium Borate - buffer(LB) around 8.6
23. Supporting medium
Supporting medium is a matrix in which the
protein separation takes place.
Various type has been used for the separation
either on slab or capillary form.
Separation is based on charge and mass of
protein,depending on the pore size of the
medium.
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24. Chemical nature Inert
Availability easy
Electrical conductivity high
Adsorptivity low
Sievingeffect desirable
Porosity controlled
Transparency high
Electro-endosmosis(EEO) low
Preservation feasible
Toxicity low
Preparation easy
25. - Starch gel
- Cellulose acetate
- Agarose
- Polyacrylamide gel
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28. Filter paper such as Whatmann no1 and no 3mm in strip of 3 or 5cm
wide have been used.
Separation takes place in 12 to 14hrs.
ADVANTAGES:
It is economical.
Easy to use.
DISADVANTAGES:
Certain compounds such as proteins, hydrophilic molecules cannot be
resolved properly due to the adsorptive and ionogenic properties of paper
which results in tailing and distortion of component bands.
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Electro osmosis.
29. 2
Separation is brought about through molecular sieving technique,
based on the molecular size of the substances. Gel material acts
as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose
gel, Starch, Polyacrylamide gels.
Gel electrophoresis
30. Agarose Gel
A linear polysaccharide (made-up of repeat unit of
agarobiose-alternating unit of galactose and 3,6-
anhydrogalactose).
Used in conc as 1% and 3%.
The gelling property are attributed to both inter- and
intramolecular hydrogen bonding
Pore size is controlled by the % of agarose used.
Large pore size are formed with lower conc and vice versa.
Purity of the agarose is based on the number of sulphate
conc, lower the conc of sulphate higher is the purity of
agarose.30
31. Easy to prepare and small
concentration of agar is required.
Resolution is superior to that of
filter paper.
Large quantities of proteins can be
separated and recovered.
It adsorbs proteins relatively less
when compared to other medium.
Sharp zones are obtained due to less
adsorption.
Resolution is less compared
to polyacrylamide gels.
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Different sources and batches of
agar tend to give different
results and purification is often
necessary.
APPLICATION:
To separate DNA, proteins, Hb variants, iso-enzymes etc.
DISADVANTAGES:ADVANTAGES:
43. POLYACRYLAMIDE GEL ELECTROPHORESIS
Frequently referred to as PAGE.
Cross-linked polyacrylamide gel are formed from the
polymerization of the monomer in presence of small amount
of N,N”-methylene- bisacrylamide.
Bisacrylamide – two acrylamide linked by the methylene group.
The polymerization of the acrylamide is an example for free
radical catalysis.
Made in conc. between 3-30% acrylamide.
low % has large pore size and vice versa.
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44.
45. SDS-PAGE
Sodium dodecyl sulphate- polyacrylamide
gel electrophoresis.
Most widely used method for analysing protein
mixture qualitatively.
Useful for monitoring protein purification – as separation
of protein is based on the size of the particle.
Can also be used for determining the relative molecular
mass of a protein.
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46. Mercaptoethanolwill breakthe disulphide bridges.
SDSbindsstronglyto anddenatures the protein.
Eachprotein isfullydenatured andopen into rod-shape with
series ofnegativelychargedSDSmoleculeon polypeptide chain.
SDSisananionic
detergent.
The sample is first
boiled for 5min in
buffercontaining
• Beta-
Mercaptoethanol
• SDS
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47. On average, One SDS molecule bind for every two
amino acid residue.
Hence original native charge is completely swamped by
the negative charge of SDS molecule.
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52. Stacking gel:
ordering/arranging and conc. the macromolecule before
entering the fieldofseparation. (4% of acrylamide)
Purpose is to concentrate protein sample in sharp band
before it enters main separating gel.
Running gel:
the actual zone of separation of the particle/molecules based
on their mobility.(15% of acrylamide)
57. - Research tool
- Measuringmolecular weight
- Peptide mapping
- Protein identification
- Determination of sample purity
- Separation of proteins and
establishing size
- Blotting
- Smaller fragments of DNA
APPLICATION:
58. ADVANTAGES:
-Gels are stable over wide range of pH and
temperature.
-Gels of different pore size can be formed.
-Simple and separation speed is good comparatively.
-Exhibitreasonablemechanicalstrength
-Lowendosmosis effect
DISADVANTAGES
-Gel preparation andcasting is time-consuming
-carcinogenic
-Complete reproducibility ofgelpreparation not possible
59. STAINING:
Fluorescentstains- Ethidiumbromide – Nucleic acids
Silverstainfor protein gel(sensitive50 times dyebased)
Dyebased– Coomassie blue–protein band
Trackingdyes– BPB>xylenecyanol
60. Native(buffer) gel
Done byusingthe polyacrylamidegel(7.5%).
SDSis absent.
pHof 8.7
Proteins are separated accordingto the electrophoretic
mobility &Sievingeffect ofthe gel.
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61. Isoelectric focussing
First described by- H.Svensson in Sweden.
Method is ideal for the separation of the
amphoteric substances.
Method has high resolution.
Able to separate the protein which differ in isoelectric
point by as little as little 0.01 of pH unit.
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63. Establishmentofph gradient:
This is achieved by the ampholyte & must have following
prop:
Must dictate pH course (buffering capacity at their pI)
Should have conductance at their pI.
Low molecular weight
Soluble in water
Low light absorbance at 280nm.
Available commercially with pH band (3-11)
Eg: Ampholine, Pharmalyte and Bio-lyte.63
68. This combination gives sophisticated analytical method
for analysing the protein mixture.
Size very from 20*20cm to the minigel.
IEF is carried on acrylamide gel (18cm*3mm), with
8M urea.
After separation, placed on 10% SDS-PAGE for further
separation .
Usedinfieldof proteomics.
Canseparate1000 to 3000 proteins fromthe cellor tissueextract.
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70. Isotachophoresis
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Used for separation of smaller ionic substances.
They migrate adjacent in contact with one another, but not
overlapping.
The sample is not mixed with the buffer prior to run.
Hence current flow is carried entirely by the sample ions.
Faster moving ions migrate first and the adjacent ones next
with no gap between the zone .
71. Allionsmigrate at the rate offastestion in zones.
Thenit ismeasuredbyUVabsorbance.
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Application-
Separationofsmallanionsand cations
Aminoacids
Peptides
Nucleotides
Nucleosides
Proteins.
72. Pulsed-Field Electrophoresis
Powerisappliedalternativelyto different pair of electrodes
Electrophoretic fieldiscycledat 105-1800
Becauseofwhichthe moleculehaveto orient to the newfield direction
Thispermit separation oflargemoleculelike DNA.
Applied:for typingvariousstrains of DNA.
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73.
74. Immunoelectrophoresis refers to precipitation in agar under an
electric field.
It is a process of combination of immuno-diffusion and
electrophoresis.
An antigen mixture is first separated into its component parts by
electrophoresis and then tested by double immuno-diffusion.
Antigens are placed into wells cut in a gel (without antibody) and
electrophoresed. A trough is then cut in the gel into which
antibodies are placed.
The antibodies diffuse laterally to meet diffusing antigen, and
lattice formation and precipitation occur permitting determination
of the nature of the antigens.
The term “immunoelectrophoresis” was first coined by Grabar and
Williams in 1953.
Immuno electrophoresis
75. Principle
When electric current is applied to a slide layered with
gel, antigen mixture placed in wells is separated into
individual antigen components according to their
charge and size. Following electrophoresis, the
separated antigens are reacted with specific antisera
placed in troughs parallel to the electrophoretic
migration and diffusion is allowed to occur. Antiserum
present in the trough moves toward the antigen
components resulting in formation of separate
precipitin lines in 18-24 hrs, each indicating reaction
between individual proteins with its antibody.
76. -Precipitation reactions are basedon the interaction of antibodies and antigens. They
are basedon two soluble reactants that come together to makeone insoluble
product, the precipitate (Figure).
-Thesereactions depend on the formation of lattices (cross-links) when antigen and
antibody exist in optimal proportions.[ it is known aszoneofequivalence and appears
to usas precipitation].
-Excessof either component reduces lattice formation and subsequentprecipitation.
PrecipitationReactions:
77.
78.
79. Procedure
Agarose gel is prepared on a glass slide put in a horizontal position.
Using sample template, wells are borne on the application zone carefully.
The sample is diluted 2:3 with protein diluent solution (20μl antigen solution
+10 μl diluent).
Using a 5 μl pipette, 5 μl of control and sample is applied across each
corresponding slit (Control slit and Sample slit).
The gel is placed into the electrophoresis chamber with the samples on the
cathode side, and electrophoresis run for 20 mins/ 100 volts.
After electrophoresis completes, 20 μl of the corresponding antiserum is added
to troughs in moist chamber and incubated for 18- 20 hours at room
temperature on a horizontal position.
The agarose gel is placed on a horizontal position, and dried with blotter
sheets.
The gel in saline solution is soaked for 10 minutes and the drying and washing
repeated twice again.
The gel is dried at a temperature less than 70°C and may be stained with
80. Results
o Presence of elliptical precipitin arcs represents antigen-
antibody interaction.
o Absence of formation of precipitate suggests no
reaction.
o Different antigens (proteins) can be identified based on
the intensity, shape, and position of the precipitation
lines.
81. Applications
The test helps in identification and approximate quantization of
various antigens in immunology.
suspected monoclonal and polyclonal gammopathies.
Used to analyze complex protein mixtures containing different
antigens.
The medical diagnostic use is of value where certain proteins are
suspected of being absent (e.g., hypogammaglobulinemia) or
overproduced (e.g., multiple myeloma).
This method is useful to monitor antigen and antigen-antibody
purity and to identify a single antigen in a mixture of antigens.
82. 50μm – ID.
300 μm – ED.
Length– 50-100cm.
Fusedsilicacapillary tube.
Polyimidecoating external.
Packedwith the buffer in use.
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oTechnique first described by- Jorgensen and Lukas (1980’s). As the
name suggest, the separation is carried in a narrow bore Capillary
Capillary electrophoresis
84. Sampleapplicationisdone byeither ofone method
Highvoltage
injection
High voltageinjection
Thebufferreservoir is
replaced bythe sample
reservoir the high
voltageisapplied(+
electrode) buffer
reservoir isplacedagain
andvoltageappliedfor the
separation.
Pressure
injection
Pressure injection
Anodicend ofcapillary is
removedfrom bufferand
placedin air tight sample
sol with pressure sample
ispushedinto capillary
kept backin the
buffersampleandvoltage
is applied.
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85. High voltage is applied (up to 50 kV)
The components migrate at different rate along the length.
Although separated by the electrophoretic migration, all the
sample is drawn towards cathode by electroendosmosis.
Since this flow is strong, the rate of electro endosmotic flow is
greater than the Electrophoretic velocity of the analyte ion,
regardless of the charge.
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88. Troubleshooting :
Adsorption of protein to the wall of capillary – leading to
smearing of protein – viewed as peak broadening – or
complete loss of protein.
- Use of neutral coating group to the inner surface of
the capillary.
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92. Instrumentation
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Similarto the capillary electrophoresis.
Separationchannel
Sampleinjection (50-100pL)
Reservoirs
Voltage(1-4kV)
samplepreparation
Pre column or post column reactors.
ClassicalCross-T design.
Timeperiod of 50-200sec.
