Serum Electrophoresis

Electrophoresis

• the Margination of charged solutes or particles in an electric field

• Migration with electricity

• Separates proteins on the basis of their electric charge densities, determined by the pH of their surrounding buffer

Arne Wilhelm Kaurin Tiselius

• Swedish biochemist

• Won Nobel Price in chemistry

• He separated proteins dissolved in an electrolyte solution by application of an electric current through a U-shaped quartz tube that held the protein solution.

Moving Boundary or Frontal Electrophoresis

What was the term of the technique of protein separation by Arne Wilhelm Kaurin Tiselius ?

It separates proteins on the basis of their electric charged densities

How does Electrophoresis work?

Cathode

Negative Pole

Anode

Positive Pole

Their charge density, which is influenced by the pH of the surrounding buffer.

What determines the movement of proteins in an electric current?

Positively charged protein

What is a cation in protein electrophoresis?

Negatively charged protein

What is an anion in protein electrophoresis?

Negative pole

Toward which pole do cations migrate?

Positive pole

Toward which pole do anions migrate?

Oppositely charged electrodes

What are charged particles attracted to in electrophoresis?

1. Net charge of the particle

2. Size of the particle

3. Shape of the particle

4. Strength of the electric field

5. Chemical and physical properties of the supporting

6. electrophoretic medium

List the factors that control the rate of protein migration in electrophoresis.

Net charge of the particle

The rate of migration is directly proportional to what?

• Size of the particle

• Viscosity of the buffer

The rate of migration is inversely proportional to what two factors?

Net charge of the particle

What is the most important factor affecting protein migration?

Iontophoresis

• Refers to the migration of small ions

• A transdermal delivery system that uses electrophoresis to the skin by a low electrical current

Hyperhidrosis

What condition is commonly treated using iontophoresis?

Zone Electrophoresis

• Usually used in clinical laboratories

• Is the migration of charged macromolecules in a porous support medium.

• paper

• Cellulose acetate (commonly used)

• Agarose gel film (DNA analysis)

What are the porous support medium for Zone Electrophoresis?

Electrophoretogram

• is the result of zone electrophoresis

• A visualization of the migration of proteins

• Consists of sharply separated zones of a macromolecule

Smaller molecules

It terms of size , these molecules will migrate faster because they are lighter

Globular proteins

In terms of shape, these proteins migrate faster than fibrous proteins.

• they have a more compact spherical shape leading to greater mobility.

Fibrous proteins

These proteins have long, rod-like structure that hinders movement

Support medium

Is a porous matrix that serves as the stationary phase for separating molecules like DNA, RNA, or proteins

• Agarose gel

• Polyacrylamide gel

• Cellulose acetate

Give the different kinds/types of support medium

Agarose gel

A support medium that has neutral charge

Polyacrylamide gel

A support medium that is porous.

Electrical charge

Basis of separation and Migration of Agarose gel

Molecular weight or Size of molecules

Basis of separation and Migration of Cellulose acetate and Polyacrylamide gel

cluster around a migrating particle

What is the action of the ions during electrophoresis?

• The higher the size of the ionic cloud

• Lower the mobility of the particle

Complete the sentence:

Ionic Strength

• The higher the ionic concentration , ______________________and the __________________.

It produces sharper protein-band separation

What effect does greater ionic strength have on protein-band separation?

It causes increased heat production

What is a disadvantage of increased ionic strength in electrophoresis?

denature heat-labile proteins

What is the consequence of excessive heat during electrophoresis?

Bands become broader instead of sharp

What happens to protein bands when heat denaturation occurs?

• False.

• Although it may sharpen bands, it also increases heat, which can damage proteins.

True or False:

• Increasing ionic strength always improves electrophoresis results.

• Why?

Ampholyte binds with H+ , becomes positively charged , and migrates toward the cathode

What happens if the buffer is more than the isoelectric point (pl) of the ampholyte?

Ampholyte loses H+ , becomes negatively charged, migrate towards the anode

What happens if the buffer is more basic than the isoelectric point (pl)?

5 minutes

How long is the sample soaked in hydrated support ?

Buffer

What is added to the chamber to maintain contact with the support?

50 V - 200 V

Electrophoresis is carried out by applying a constant voltage of ?

• Driving force

• Support medium

• Buffer

• Sample

• Detecting system

Components of Electrophoresis

• pH

• Ionic strength

Two buffer properties that affect the charge of Ampholytes

8.6

pH of Cellulose Acetate gel

Ampholyte

Is a molecule , such as protein, whose net charge can be either positive or negative

Paper electrophoresis

Has been replaced by cellulose acetate or agarose gel in clinical laboratories

Acetic anhydride

Cellulose is acetylated to form cellulose acetate by treating it with ?

Cellulose acetate

A dry, brittle film composed of about 80% air space, is produced commercially

Can be made Transparent

Advantage of cellulose acetate

Agarose gel

Used as a purified fraction of agar, it is neutral and, therefore,does not produce electroendosmosis.

2mL

How many mL of sample does Agarose gel require?

Seaweeds

Where does Agarose gel come from?

Polyacrylamide gel

• separates serum proteins into 20 or more fractions

• Widely used to study individual proteins

Going Down

Direction of migration for Polyacrylamide gel

Starch Gel

• Not widely used because of technical difficulty in preparing the gel

• Used for research and never in clinical laboratory

Surface charge and molecular size

Starch gel basis of separation ?

Greater uniformity

Advantage of Starch gel

Electroendosmosis

The movement of buffer ions and solvent relative to the fixed support

• Negative charge

• Hydroxyl ions

In Electroendosmosis, Support media (paper, cellulose acetate, and agar gel) take on a _________ charge from absorption of ___________.

• hydroxyl ions

• Free positive ions

When current is applied to the electrophoresis system, the _________ remained fixed, while the ___________ move toward the cathode.

They are swept toward the cathode with the solvent

What happens to molecules that are nearly neutral?

True

True or False:

• Support media such as agarose and acrylamide gel are essentially neutral, eliminating electroendosmosis.

The support medium should have electric neutrality

How to achieve optimum separation of proteins?

Serum

What is the preferred specimen?

Plasma

This specimen should be avoided because fibrinogen will appear as a distinct narrow band between the beta and gamma fractions.

M spike

Using Plasma specimen can be mistaken as ?

Monoclonal gammopathy

Appearance of fibrinogen is important because it is used to detect?

Cerebrospinal fluid

This specimen can be used if concentrated up to 300 times, depending on the original protein concentration.

Hemoglobin hemolysate

Is used without further concentration

2-5 mL

Cellulose acetate and Agarose gel electrophoresis require approximately volume range of sample?

Twin-wire applicator

Designed to transfer a small amount of sample

Densitometry

Most common and reliable way for quantitation

• measurement of the density of light passing through the fraction

Stained

Separated protein fractions are ______ to reveal their locations?

• Amido black

• Ponceau S

• Coomassie blue

What are the stains used in Detection and quantitation?

Comassie blue

stain used for Urine and CSF

Silver staining

This stain is used in cases where the protein concentrations are low

Identify the parts of the densitometer

Filters

This part of the densitometer permit only a certain wavelength range to spike the strip.

• Isoelectric Focusing

• High-Resolution Protein Electrophoresis

Types of Electrophoresis

Isoelectric Focusing

• A modification of electrophoresis

• Charged proteins migrate through a support medium that has a continuous pH gradient.

Isoelectric point

Isoelectric focusing exploits a different parameter associated with protein charges. What parameter is this?

Isoelectric point

For each protein molecule, there is a pH where the net charge on the molecule is zero.

High-Resolution Protein Electrophoresis

Uses a higher voltage coupled with a cooling system in the electrophoretic apparatus and a more concentrated buffer.

Agarose buffer

Most commonly used medium for High-Resolution Protein Electrophoresis

Prealbumin

What protein is present in zone 1?

Albumin

What protein is present in zone 2?

a-Lipoprotein (a-Fetoprotein)

What protein is present in zone 3?

a1 - Antitrypsin , a1 - Acid glycoprotein

What protein is present in zone 4 ?

Gc - globulin , inter-a-trypsin inhibitor, a1 - antichymotrypsin

What protein is present in zone 5 ?

a2 - Macroglobulin , Haptoglobin

What protein is present in zone 6 ?

Cold insoluble globulin, (Hemoglobin)

What protein is present in zone 7 ?

Transferrin

What protein is present in zone 8 ?

B - Lipoprotein

What protein is present in zone 9 ?

C3

What protein is present in zone 10 ?

IgA (Fibrinogen) ,

IgM (Monoclonal Igs, light chain)

What protein is present in zone 11 ?

IgC (C- reactive protein)

What protein is present in zone 12 ?

Serum Electrophoresis

Serum samples are applied close to the cathode end of a support medium that is saturated with an alkaline buffer . (ph 8.6)

Cathodes and Anodes

Electrode that will be used to separate the protein

True

True or False:

• All major proteins carry a net negative charge at pH 8.6 and migrate toward the anode