CLS 414 Clinical Chemistry Student Lab Rotation:

Basic Principles of Electrophoresis Lecture 1

Basic Principles of
Electrophoresis
Universityof Nebraska Medical Center
p
Ricki Otten MT(ASCP)SC
uotten@unmc.edu
Electrophoresis
Electrophoresis is a separation technique
Technique is used in clinical laboratories to
t t i f h th
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separate proteins from each other:
Proteins in body fluids: serum, urine, CSF
Proteins in erythrocytes: hemoglobin
Nucleic acids: DNA, RNA
Basic Terms
Amphoteric nature of proteins
Zwitterion
Isoelectric point (pI)
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The overall charge of the protein is determined by
the number of acidic and basic amino acids in its
basic structure. Because of their amphoteric
nature, amino acids can express a net positive
charge, a net negative charge or a net charge of
zero.
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Net Charge of Molecule
pH of the buffer (reagent) determines the
charge of the molecule
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Net Charge of Molecule
Net charge of molecule determines
migration direction in electrical field
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Cathode Anode
(Negative electrode) (Positive electrode)
CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 2
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
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pI and Zwitterion
pI (pH) where molecule remains neutral
Will not migrate in an electrical field
Remains at application point
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(cathode -) Net zero charge (anode +)
(will not migrate)
Every amino acid has its own specific
isoelectric point. Since proteins are made of
amino acids, all proteins have their own pI
Pre-albumin: pI ~pH 4.7
Albumin: pI ~pH 4 9
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Albumin: pI pH 4.9
Gamma globulins: pI ~pH 7.3
How charged a molecule becomes depends
on the pH of the buffer and the proteins
isoelectric point
At a pH above its isoelectric point, the proteins will have
a net negative charge and will migrate towards the anode
Since pre-albumins isoelectric point (4.7) is the furthest from
the buffer pH, it is expected to have the greatest charge and
migrate fastest towards the anode
Since the gamma globulins isoelectric point (7.3) is the
closest to the buffer pH, it is expected to have the least
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charge and migrate slowest towards the anode
Electrophoresis is a separation
technique based on the principle
that a charged particle in solution
will migrate towards one of the
electrodes when placed in an
electrical field
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electrical field
The speed and direction a
charged particle moves is
determined by the particles:
Net charge (determined by buffer pH)
Incr charge =faster speed
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Incr charge faster speed
CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 3
The speed and direction a
charged particle moves is
determined by the particles:
Size and shape
Incr size =slower speed
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The speed and direction a charged
particle moves is also influenced by
external factors such as:
Voltage
Incr voltage incr speed incr heat
protein denaturation
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protein denaturation
The speed and direction a charged
particle moves is also influenced by
external factors such as:
Buffer pH
Determines net charge of protein
and therefore direction of migration
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The speed and direction a
charged particle moves is also
influenced by external factors
such as:
Support medium (type of gel)
P t i i t ti l d
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Protein interaction slows speed
The speed and direction a charged
particle moves is also influenced by
external factors such as:
Temperature
Incr temp Incr speed incr heat
leads to denaturation
D t d d
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Decr temp decr speed
The proteins found in plasma (TSP) all have amino acids as
their subunits, and each protein has its own specific
isoelectric point
Because of their different isoelectric points, each protein will
move at a different rate when placed in an electrical field
Proteins with similar isoelectric points will migrate to a similar
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area in an electrical field
CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 4
Recall that total serum protein (TSP) is comprised
of albumin and globulins.
Electrophoresis separates TSP into 5 distinct
zones or bands:
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The width of each band is dependent upon the
number of proteins that are present in that
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:
Transferrin, Complement, beta-
( )
alpha-2-macroglobulin,
Haptoglobin, Ceruloplasmin
Thyroxine-binding globulin,
alpha-1-antitrypsin,
alpha-1-lipoprotein (HDL)
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IgG, IgA, IgM, IgD, IgE
and C-reactive protein
Lipoprotein (LDL)
Basic Procedure
1.Sample is applied to an agarose gel
2.Gel is placed into electrophoresis cell
containing barbital buffer at pH 8.6
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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
6.Gel and densitometer scan are
l t d
Instrumentation and Reagents
Electrophoresis cell
2 compartment cell
Buffer
2platinumelectrodes
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2 platinum electrodes
Anode
Cathode
Negative Electrode =Cathode
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CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 5
Instrumentation and Reagents
Power source
Buffer: barbital, pH 8.6
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Carries applied current
Determines charge
and migration direction
Fill Both Compartments of
Cell with Buffer
Procedure
manual
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Instrumentation and Reagents
Support media
Various types
Minimize interactions:
pure and neutral
Agarose: often used
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Agarose: often used
Electroendosmosis
effects minimal
Clarity: scanning
possible
Commercial prep
Miniaturization
Native Clarity After Drying
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Instrumentation and Reagents
Fixative, Stain and Rinse solutions
Fix proteins to gel surface
Stain proteins to visualize
P t i t i
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Protein stain
Lipid (fat) stain
Nucleic acid stain
Excess stain rinsed away
Fixative, Stain and De-stain
Solutions: Corrosive
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CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 6
Stained Gel
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De-stained Gel
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Instrumentation and Reagents
Drying oven
De-stained gel is dried
Clear gel ready to scan using densitometer
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Densitometer
A densitometer is a
special type of
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special type of
spectrophotometer used
to measure light
transmittance through a
solid sample such as
an electrophoretic strip
Densitometer
The electrophoretic
strip is moved past
a measuring optical
system.
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The absorbance of
each band is
measured and the
area of each fraction
is displayed on a
strip chart recorder
Densitometer
Each peak represents an individual band on the
electrophoretic strip
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CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 7
Densitometer
Quantitation is performed by determining
the area of each band as a percent of the
total area for that scan
Microprocessors
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Microprocessors
automatically integrate
and compute the area
under each peak and
present the data in both
percent and
concentration units
Albumin
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Ig
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Parameters Affecting
Electrophoresis
pH
Ionic strength of buffer
Ions present
Current
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Current
Voltage
Temperature
Time
Medium
Technical Considerations
Buffers
Barbital bacterial growth pH change
Barbital, pH 8.6 most often used
Discard after each run
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Sample
Optimal amount of sample applied to gel
Avoid overloading: dilute serum prior to
application (0.050 ml serum +0.2 ml buffer)
Pop Quiz !
What is the dilution?
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0.050 ml serum + 0.2 ml buffer
Pop Quiz !
What is the dilution?
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0.050 ml serum + 0.2 ml buffer
0.050 + 0.200 = 0.250 total
0.050 : 0.250
1 : 5
CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 8
Technical Considerations
Evaporation and wick flow
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Electroendosmosis
Electroendosmosis
Surface of gel is negatively charged
Surface gel ions are immobile
Positive buffer ions (pH 8.6) orient with
negative surface ions =positive ionic cloud
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negative surface ions positive ionic cloud
Electroendosmosis
Ionic cloud is mobile
Electrical current causes positive ionic cloud to
move toward the cathode
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Electroendosmosis
Molecules on surface of gel that hold a weak
negative charge are pushed toward the cathode
despite migration direction toward the anode
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Electroendosmosis
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
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Types of Electrophoresis
Agarose, cellulose, polyacrylamide
Iso-electric focusing
Counter-current electrophoresis
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Two-dimensional electrophoresis
High resolution electrophoresis
Capillary electrophoresis
CLS 414 Clinical Chemistry Student Lab Rotation:
Basic Principles of Electrophoresis Lecture 9
Blotting Techniques
General procedure
Separation by electrophoresis
Separated components transferred (blotted)
to a specific membrane (nylon cellulose gel)
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to a specific membrane (nylon, cellulose, gel)
Detected using nucleic acid probe
Southern blot: DNA, DNA fragments
Northern blot: RNA, RNA fragments
Western blot: viral antibodies (HIV-1)
Electrophoresis is a technique used in
clinical laboratories to separate particles
(proteins) from each other:
Proteins in body fluids: serum, urine, CSF
Proteins in erythrocytes: hemoglobin
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Nucleic acids: DNA, RNA