clinical chemistry

1. ELECTROPHORESIS
BASIC CONCEPTS,
INSTRUMENTATION, TECHNIQUE,
TYPES AND APPLICATIONS
By
Dr. Basil, B – MBBS (Nigeria),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi.
February, 2015.

2. INTRODUCTION
• ELECTROPHORESIS – migration of charged solutes
or particles in a liquid medium under the influence
of an electric field
• The aim of carrying out electrophoresis include:
– To determine the number, amount and mobility of
components in a given sample or to separate them.
– To obtain information about the electrical double layers
surrounding the particles.
– Determination of molecular weight of proteins and DNA
sequencing.

3. BASIC CONCEPTS
• Iontophoresis – migration of small ions
• Ionized species move towards the anode or cathode
depending on their charges
• Ampholytes become +vely charged in a soln more
acidic than its isoelectric point (pI), and –vely charged
in a more alkaline soln
• Rate of migration depends on:
– Net electrical charge of the molecule
– Size and shape of the molecule
– Electrical field strength
– Properties of the supporting medium
– Temperature of operation

4. • Electrophoretic mobility (µ) – rate of migration (cm/s)
per unit field strength (volts/cm)
µ = Q/6∏rȠ
Where:
µ = electrophoretic mobility in cm2/(V)(s)
Q = net charge on the ion
r = ionic radius of the solute
Ƞ = viscosity of the buffer soln
• Other factors affecting mobility:
– Endosmotic flow: This is the preferential movement of
water in one direction through an electrophoresis medium
due to selective binding of one type of charge on the
surface of the medium
– Wick flow: Movement of water from the buffer reservoir
towards the center of an electrophoresis gel or strip to
replace water lost by evaporation

5. INSTRUMENTATION/REAGENTS

6. Power Supply:
• Electrophoresis is done under conditions of
constant voltage, current or power.
• Current (I) flow through the medium > heat
production due to resistance (R) > increase in
thermal agitation of dissolved ions > decrease in
resistance and increase in current flow >> more
heat and evaporation of water from the buffer >
increase in ionic concentration of the buffer
• Migration rate is kept constant by using a constant-
current power supply.
• Since, EMF = IR, and increase in R will result in and
increase in EMF (V) at constant current (I), thus, no
appreciable heat production

7. Buffers:
• Function include:
– Carries the applied current
– Establishes the pH of the system
– Determines the electrical charge on the solute
• The ionic strength influences:
– Conductance of the support
– Thickness of the ionic cloud (buffer and non-buffer ions)
surrounding a charged molecule
– Rate of its migration
– Sharpness of the electrophoretic zone
• Features of an ideal buffer:
– Does not interfere with the ability to detect the analytes of
interest
– Maintains solubility of the analytes
– Maintains buffering capacity through the analysis
– Produce the desired separation

8. • Commonly used buffers include:
– Low pH (acidic): phosphate, acetate, formate, citrate
– High pH (basic): tris, tricine, borate, CAPS (N-cyclohexyl-3-
aminopropanesulfonic acid)
• Higher the ionic strength (and concentration) > higher
size of ionic cloud > lower mobility of the particle
• Also, higher ionic strength > sharper protein-band
separation and increased heat production > denaturation
of heat-labile proteins
• Buffers used are made of monovalent ions because their
valencies (ionic strength) and molality are equal.
• They are good culture media for microorganisms and
should be refrigerated when not in use,
• Also cold buffer reduces evaporation and improves
resolution
• Small volumes should be discarded after use, but large
volumes can be reused up to 4 times

9. Support Media:
• Insoluble gels e.g sheets, slabs or columns of starch,
agarose or polyacrylamide
• Membranes (paper) of cellulose acetate
• Pure buffer soln in a capillary
Starch Gel:
• Separate macromolecules on basis of surface charge and
molecular size
• Obsolete because preparation of reproducible starch gel
is difficult
Cellulose Acetate:
• made by treating cellulose with acetic anhydride
• They need to be soaked in buffer to soften them before
use
• Require clearing before densitometry
• Hardly used in routine clinical application

10. Agarose:
• Used in AGE for the separation of Serum, Urine or
CSF proteins, Hb variants, Isoenzymes,
Lipoproteins etc.
• Separation is based only on charge-to-mass ratio –
pore size is large enough for all proteins to pass
• Separate proteins into only 5 fractions (zones):
albumin, alpha-1, alpha-2, beta- and gamma-
globulins
• Advantages:
– Permits excellent densitometry – lower affinity for
proteins (migration is not affected) and native clarity
after drying
– Little Endosmosis – free of ionizable groups (neutral)
• Disadvantage:
– DNA recovery is affected by inhibitors

11. Polyacrylamide:
• thermostable, transparent, durable and relatively
chemically inert
• No endosmosis – uncharged
• Pore size does not allow larger proteins like fibrinogen, B1-
lipoprotiens, Y-globulins etc, to migrate
• Separation is based on both charge-to-mass ratio and
molecular mass – molecular sieving
• Carcinogenic – CAUTION when handling
• Accommodates a large amount of sample in a single
sample slot
• DNA recovered is pure with no inhibitors unlike Agarose.
• Separates proteins into 20 or more fractions
• Used to study individual proteins (e.g Isoenzymes)

12. Equipment used for Gel Electrophoresis:

13. TECHNIQUE
Separation:
• Blot the hydrated support medium to remove
excess buffer
• The sample is added to the support for about 5min
• The support is then placed into the electrophoretic
chamber in contact with the buffer
• Apply a constant-current or constant-voltage
power for a specified time
• The support is then removed and placed in a
fixative or rapidly dried to prevent diffusion of the
sample

14. Staining:
• The support is then stained and dried after
washing out the excess dye
• Amido Black B or members of the Coomassie
Brillant Blue series are the commonest dyes
• The amount of dye taken up is dependent on the
type of protein, degree of denaturation by the
fixing agent and quality of the dye
• To visualize isoenzymes, incubate the gel in contact
with a solution of substrate, which is linked
structurally or chemically to a dye, before fixing.
• Typical stain solution may be used several times
• When the stain solution becomes faulty, protein
zones will appear too lightly stained
• It must be stored tightly covered to prevent
evaporation

15. Detection and Quantification:
• Detection can be achieved using UV light, but
quantification is by Densitometry
• Densitometers integrate the area under a peak, and
the result is printed as percentage of the total
• Reliable quantification of stained zones using
densitometry requires:
– Light of an appropriate wavelength
– Linear response from the instrument
– Transparent background in the strip being scanned
• Mass spectrometers – determine the molecular
weights of proteins and their cleavage products, and
for peptide sequencing.

16. Blotting Techniques:
• Southern Blotting – widely used in molecular biology:
– DNA or DNA fragments separated by AGE
– Strip of nitrocellulose or a nylon membrane is laid over the
gel blotting the DNA or DNA fragments onto it by capillary,
electro-, or vacuum blotting
– Detection and identification is by hybridization with a
labeled complementary nucleic acid probe.
• Northern Blotting:
– Separates and detects RNAs
– Uses labeled RNA probe for hybridization
• Western Blotting:
– Separates and detects proteins
– The membrane is reacted with a reagent containing an
antibody raised against the protein of interest

17. Common Problems Encountered:

18. TYPES OF ELECTROPHORESIS
• Zone Electrophoresis
• Slab Gel Electrophoresis
• Disc Electrophoresis
• Isoelectric Focusing Electrophoresis
• 2-Dimensional Electrophoresis
• Capillary Electrophoresis
• Microchip Electrophoresis

19. Zone Electrophoresis:
• Produce zones of proteins that are heterogeneous
and physically separated from one another
• Classified according to type and structure of the
support material e.g AGE, CAE, PAGE etc

20. Slab Gel Electrophoresis:
• Use of a rectangular gel regardless of the thickness
• Main advantage – ability to simultaneously separate
several samples in one run
• Primary method used in clinical chemistry lab
• Gels (usually agarose) may be cast on sheet of plastic
backing or completely encased within a plastic walled
cell allowing horizontal or vertical electrophoresis
and submersion for cooling, if needed.
• May be cast with additives like:
– Ampholytes which create a pH gradient, or
– Sodium dodecyl sulfate (SDS) that denatures protiens

21. Schematic representation of Slab gel electrophoresis

22. • Useful in the separation of serum proteins, isoenzymes,
lipoproteins, hemoglobins, and fragments of DNA and RNA
Common problems encountered in SGE include:
• Discontinuities in sample application – dirty applicator;
cleaned by agitating in water
• Unequal migration of samples across the width of the gel
– dirty electrodes (uneven application of electric field),
uneven wetting of the gel
• Distorted protein zones – bent applicator, incorporation of
air bubble during sample application, over application of
sample, excessive drying of support
• Unusual bands – artefacts, other causes e.g hemolysed
samples > increased b-globulin band
• Atypical bands – denatured protein from deteriorated
serum, also rule out a true paraprotein

23. Disc Electrophoresis:
• 3-gel system:
– small-pore separating gel (running gel),
– a larger-pore spacer gel (stacking gel),
– and a thin layer of large-pore monomer solution (sample
gel) – containing about 3 µL of serum
• The different composition cause discontinuities in
the electrophoresis matrix
• During electrophoresis, all proteins migrate easily
through the large-pore gels and stack up on the
separation gel in a very thin zone
• This improves resolution and concentrates protein
components at the border (or starting zone)
• Separation occurs at the bottom separation gel by
the molecular sieve phenomenon

24. Schematic representation of Disc Electrophoresis

25. Isoelectric Focusing Electrophoresis:
• Separates amphoteric compounds with increased
resolution in a medium possessing a stable pH gradient
• The protein migrate to a zone in the medium where the pH
of the gel matches its pI
• At this point, the charge of the protein becomes zero and
its migration ceases – it become “focused”.
• Regions associated with a given pH are very narrow –
enough to separate proteins that differ in their pI values by
only 0.02pH units
• A high voltage power source is needed because carrier
ampholytes are used in relatively high concentrations
• Thus, the electrophoretic matrix must be cooled
• IEF is used in neonatal screening programs to test for
variant Hb

27. 2-Dimensional Electrophoresis:
• Uses charge-dependent IEF (first dimension) and molecular
weight-dependent electrophoresis (in the second)
• 1st dimension – carried out in a large-pore medium like
agarose or large-pore PAG; to which ampholytes are added
to yield a pH gradient
• 2nd dimension is often polyacrylamide in a linear or
gradient format
• It achieves the highest resolving power for the separation
of DNA fragments
– 1st dimension – normal AGE
– 2nd dimension – ethidium bromide is added to the gel to open
up the fragments and cause changes in their mobility
• Method of choice when complex samples need to be
arrayed for characterization, as in proteomics.

28. Schematic representation of 2D Electrophoresis

29. Capillary Electrophoresis:
• Separation in narrow-bore fused silica capillaries (inner
diameter 25 – 75µm) filled with buffer – gel media can also
be used
• Sample is loaded after filling capillary with buffer, and
electric field applied
• Borate is a classic CE buffer that generates relatively low
current and heat, even with a high ionic strength
• Electro-osmotic flow (EOF) controls the amount of time
solutes remain in the capillary (also Electroendosmostic
flow or endosmosis)
– EOF = bulk flow of liquid towards the cathode upon application
of an electric field and it is superimposed on electrophoretic
migration
• Cations migrate fastest due to EOF and electrophoretic
attraction towards the cathode

31. • Neutral molecules are all carried by EOF but not separated
from each other
• Anions move slowest because EOF is slightly greater than
the attraction twds the anode and repulsion from cathode
• Used for determination of MWts of proteins and peptides,
analysis of PCR products, inorganic ions, organic acids,
pharmaceuticals, optic isomers, and drugs of abuse in
serum and urine.
• When using a new capillary or changing buffer, the capillary
must be equilibrated with the buffer – conditioning
– Particularly important when phosphate-containing buffer is
involved
– This should be done for at least 4hrs before electrophoresis is
started
• The capillary surface must be regenerated or reconditioned
to remove any material adsorbed onto the wall
– Done by following each run with 3- to 5-column volume rinse
using NaOH, and flushing with 5- to 8-column volumes of fresh
buffer.

32. Microchip Electrophoresis:
• Similar in principle to CE, but differs in that
the separation channels, sample injection
channels and reservoirs are all fabricated into
the same planar substrate using
photolithographic processes.
• More so, sample preparationand/or
precolumn or postcolumn reactors, detectors,
and excitation sources are intergrated into the
chip

34. QUALITY CONTROL
• A Clear SOP must be adhered to during each
run to ensure reproducibility
• Control serum must be included in each
electrophoretic run to evaluate its quality and
that of the densitometer.

35. APPLICATIONS
• DNA Analysis:
– specific DNA sequences can be analyzed, isolated and cloned.
– The analyzed DNA may be used in forensic investigations and
paternity tests.
• Protein Analysis:
– In the diagnosis of conditions where levels of specific proteins
or total protein is low or higher than normal e.g monoclonal
gammopathy etc.
• Antibiotic Analysis:
– Synthesis of new antibiotics
– Analysis of bacteria response to antibiotics and determining
antibiotic-resistance
• Vaccine Analysis:
– Purification, processing and analysis of vaccines e.g influenza
vaccine, hepatitis vaccine, polio vaccine

36. REFERENCES:
• Tietz Textbook of Clinical Chemistry and Molecular
Diagnostics, fifth ed., by Burtis et al.
• Clinical Chemistry; Principles, Techniques and
Correlations, 7th ed., by Bishop et al.
• http://www.slideshare.net/jyots23/electrophoresis-
presentation
• http://www.bio-rad.com/en-us/applications-
technologies/protein-electrophoresis-methods
• Landers JP. Molecular diagnostics on electrophoretic
microchips. Anal Chem 2003;75:2919-27
• St. claire iii RL. Capillary electrophoresis. Anal Chem
1996;379-423

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