University of Nebraska Medical Center

Electrophoresis
• Electrophoresis is a separation technique
Basic Principles of
p
Electrophoresis • Technique is used in clinical laboratories to
separate
t proteins
t i from
f eachh other:
th
– Proteins in body fluids: serum, urine, CSF
– Proteins in erythrocytes: hemoglobin
Ricki Otten MT(ASCP)SC – Nucleic acids: DNA, RNA
uotten@unmc.edu

The overall charge of the protein is determined by

Basic Terms the number of acidic and basic amino acids in its
basic structure. Because of their amphoteric
• Amphoteric nature of proteins nature, amino acids can express a net positive
• Zwitterion charge, a net negative charge or a net charge of
• Isoelectric point (pI) zero.

3 4

Net Charge of Molecule Net Charge of Molecule

• pH of the buffer (reagent) determines the • Net charge of molecule determines
charge of the molecule migration direction in electrical field

Cathode Anode
5 (Negative electrode) (Positive electrode)
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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 1
 At one pH, called the isoelectric point (pI), the
number of positive and negative charges are equal.
At this pH, the protein exhibits a net zero charge,
and is referred to as a ‘zwitterion’
pI and Zwitterion
• pI (pH) where molecule remains neutral
• Will not migrate in an electrical field
• Remains at application point

(cathode -) Net zero charge (anode +)

(will not migrate)

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At a pH above its isoelectric point, the proteins will have

• Every amino acid has its own specific a net negative charge and will migrate towards the anode

isoelectric point. Since proteins are made of Since pre-albumin’s isoelectric point (4.7) is the ‘furthest’ from
amino acids, all proteins have their own pI the buffer pH, it is expected to have the greatest charge and
migrate ‘fastest’ towards the anode

Pre-albumin: pI ~ pH 4.7 Since the gamma globulins’ isoelectric point (7.3) is the
Albumin: pI ~ pH 4
4.9
9 ‘closest’ to the buffer pH, it is expected to have the least
charge and migrate ‘slowest’ towards the anode
Gamma globulins: pI ~ pH 7.3

• How ‘charged’ a molecule becomes depends

on the pH of the buffer and the protein’s
isoelectric point
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Electrophoresis is a separation The speed and direction a

technique based on the principle charged particle moves is
that a charged particle in solution determined by the particle’s:
will migrate towards one of the
electrodes when placed in an Net charge (determined by buffer pH)
electrical field Incr charge = ‘faster
faster speed’
speed

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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 2
 The speed and direction a The speed and direction a charged
charged particle moves is particle moves is also influenced by
determined by the particle’s: external factors such as:

Voltage
Size and shape
Incr voltage  incr speed  incr heat
Incr size = slower speed
 protein denaturation

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The speed and direction a charged The speed and direction a

particle moves is also influenced by charged particle moves is also
external factors such as: influenced by external factors
such as:
Buffer pH
Determines net charge of protein
and therefore direction of migration Support medium (type of gel)
i t ti  slows
P t i interaction
Protein l speed
d

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The speed and direction a charged The proteins found in plasma (TSP) all have amino acids as
particle moves is also influenced by their subunits, and each protein has its own specific
external factors such as:
isoelectric point

Temperature Because of their different isoelectric points, each protein will

Incr temp  Incr speed  incr heat move at a different rate when placed in an electrical field
 leads to denaturation
Proteins with similar isoelectric points will migrate to a similar
D
Decr ttemp  decr
d speed
d area in an electrical field

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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 3
 Recall that total serum protein (TSP) is comprised
of albumin and globulins. The width of each band is dependent upon the
Electrophoresis separates TSP into 5 distinct number of proteins that are present in that
zones or bands: fraction

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Of these five major fractions, 4 are composed of a number of

additional proteins of varying size and molecular weight. The
clinically significant proteins are listed:
Thyroxine-binding globulin,
alpha-1-antitrypsin,
alpha-2-macroglobulin,
alpha-1-lipoprotein (HDL)
Haptoglobin, Ceruloplasmin
Transferrin, Complement, beta-
Lipoprotein (LDL)
( )
IgG, IgA, IgM, IgD, IgE
and C-reactive protein
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Basic Procedure
• 1.Sample is applied to an agarose gel
• 2.Gel is placed into electrophoresis cell
containing barbital buffer at pH 8.6
3. Power is applied creating an electrical
field and the proteins are separated
• 4.Proteins are fixed to the gel and
stained
• 5.Separated proteins on gel are scanned
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• 6.Gel and densitometer scan are
l t d

Instrumentation and Reagents Negative Electrode = Cathode

• Electrophoresis cell
– 2 compartment cell
– Buffer
– 2 platinum electrodes
• Anode
• Cathode

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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 4
 Fill Both Compartments of
Instrumentation and Reagents
Cell with Buffer
• Power source
Procedure
manual

• Buffer: barbital, pH 8.6

– Carries applied current
– Determines charge
and migration direction

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Instrumentation and Reagents ‘Native Clarity’ After Drying

• Support media
– Various types
– Minimize interactions:
pure and neutral
• Agarose: often used
– Electroendosmosis
effects minimal
– Clarity: scanning
possible
– Commercial prep
– Miniaturization
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Fixative, Stain and De-stain

Instrumentation and Reagents
Solutions: Corrosive
• Fixative, Stain and Rinse solutions
– Fix proteins to gel surface
– Stain proteins to visualize
• P
Protein
t i stain
t i
• Lipid (fat) stain
• Nucleic acid stain
– Excess stain rinsed away

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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 5
 Stained Gel De-stained Gel

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Instrumentation and Reagents Densitometer

• Drying oven
– De-stained gel is dried
• Clear gel ready to scan using densitometer A densitometer is a
special type of
spectrophotometer used
to measure light
transmittance through a
solid sample such as
an electrophoretic strip
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Densitometer Densitometer
The electrophoretic Each peak represents an individual band on the
strip is moved past electrophoretic strip
a measuring optical
system.
The absorbance of
each band is
measured and the
area of each fraction
is displayed on a
strip chart recorder 35 36

CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 6
 n
tei
ro
op
Albumin -lip
Densitometer alp
ha
-1
d
an
Quantitation is performed by determining ro
tei
n
lin
op sin bu tei
n
the area of each band as a percent of the g lyc tryp oglo ro
-1- anti acr op C3
h a 1-
-m i n - lip errin ent
total area for that scan p -
Al pha ha-2 glob Bet ans plem
a f
Al Alp pto T r om
Ha C IgM G
IgA Ig
Microprocessors
automatically integrate
and compute the area
under each peak and
present the data in both
percent and
concentration units
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Parameters Affecting
Technical Considerations
Electrophoresis
• pH • Buffers
• Ionic strength of buffer – Barbital  bacterial growth  pH change
• Ions present – Barbital, pH 8.6 most often used
– Discard after each run
• Current
• Voltage
• Sample
• Temperature
– Optimal amount of sample applied to gel
• Time – Avoid ‘overloading’: dilute serum prior to
• Medium application (0.050 ml serum + 0.2 ml buffer)

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Pop Quiz ! Pop Quiz !

What is the dilution?
What is the dilution?

0.050 ml serum + 0.2 ml buffer 0.050 ml serum + 0.2 ml buffer

0.050 + 0.200 = 0.250 total
0.050 : 0.250
1:5
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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 7
 Technical Considerations Electroendosmosis
• Surface of gel is negatively charged
• Evaporation and wick flow
• Surface gel ions are immobile
• Positive buffer ions (pH 8.6) orient with
negative surface ions = positive ionic cloud

• Electroendosmosis
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Electroendosmosis Electroendosmosis
• Molecules on surface of gel that hold a weak
• Ionic cloud is mobile negative charge are ‘pushed’ toward the cathode
• Electrical current causes positive ionic cloud to despite migration direction toward the anode
move toward the cathode

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Electroendosmosis Types of Electrophoresis

• Macromolecules (proteins) that have a sufficiently
strong enough charge are able to oppose the flow
of the positive ion cloud and move in the opposite
direction towards the electrode of opposite polarity
• Agarose, cellulose, polyacrylamide
• Iso-electric focusing
• Counter-current electrophoresis
• Two-dimensional electrophoresis
• High resolution electrophoresis
• Capillary electrophoresis

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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 8
 Blotting Techniques • Electrophoresis is a technique used in
clinical laboratories to separate particles
• General procedure (proteins) from each other:
– Separation by electrophoresis – Proteins in body fluids: serum, urine, CSF
– Separated components transferred (blotted) – Proteins in erythrocytes: hemoglobin
to a specific membrane (nylon
(nylon, cellulose
cellulose, gel)
– Nucleic acids: DNA, RNA
– Detected using nucleic acid probe
• Southern blot: DNA, DNA fragments
• Northern blot: RNA, RNA fragments
• Western blot: viral antibodies (HIV-1)
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CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 9