CLS 414 Clinical Chemistry Student Lab Rotation: Basic Principles of Electrophoresis Lecture 1

Basic Principles of Electrophoresis

University of Nebraska Medical Center

p

Ricki Otten MT(ASCP)SCuotten@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 bythe number of acidic and basic amino acids in itsbasic structure. Because of their amphotericnature, amino acids can express a net positivecharge, a net negative charge or a net charge ofzero.

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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 specificisoelectric point. Since proteins are made ofamino acids, all proteins have their own pI

Pre-albumin: pI ~ pH 4.7Albumin: pI ~ pH 4 9

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Albumin: pI pH 4.9Gamma globulins: pI ~ pH 7.3

How charged a molecule becomes dependson the pH of the buffer and the proteinsisoelectric point

At a pH above its isoelectric point, the proteins will havea net negative charge and will migrate towards the anode

Since pre-albumins isoelectric point (4.7) is the furthest fromthe buffer pH, it is expected to have the greatest charge andmigrate fastest towards the anode

Since the gamma globulins isoelectric point (7.3) is theclosest 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 separationtechnique based on the principlethat a charged particle in solutionwill migrate towards one of theelectrodes when placed in anelectrical 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 shapeIncr size = slower speed

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The speed and direction a chargedparticle moves is also influenced byexternal factors such as:

VoltageIncr 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 pHDetermines 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 factorssuch 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:

TemperatureIncr 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 astheir subunits, and each protein has its own specificisoelectric point

Because of their different isoelectric points, each protein willmove 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 thenumber of proteins that are present in thatfraction

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Of these five major fractions, 4 are composed of a number ofadditional proteins of varying size and molecular weight. Theclinically 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 electricalfield 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

2 platinum electrodes

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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 usedto measure lighttransmittance through asolid sample such asan electrophoretic strip

DensitometerThe electrophoretic strip is moved past a measuring opticalsystem.

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The absorbance ofeach band is measured and thearea of each fractionis displayed on astrip chart recorder

DensitometerEach peak represents an individual band on theelectrophoretic strip

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CLS 414 Clinical Chemistry Student Lab Rotation: Basic Principles of Electrophoresis Lecture 7

DensitometerQuantitation is performed by determiningthe area of each band as a percent of thetotal 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

Alpha-

1-glyc

oprote

in and

alpha-

1-lipop

rotein

Alpha-

1-antit

rypsin

Alpha-

2-macr

oglobu

lin

Hapto

globin Be

ta-lipo

protein

Transf

errin

Comp

lement

C3

IgAIgM IgG

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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 buffer0.050 + 0.200 = 0.250 total

0.050 : 0.2501 : 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 oppositedirection 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 inclinical 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