Biochemical instrumental analysis-7

Dr. Maha Al-Sedik

Electrophoresis

Atoms are neutral because the protons which are positive

and electron that are negative balance out the charge, so

there are same numbers of protons and electrons in an

atom.

An ion is an atom or group of atoms that has a positive or

negative charge.

Ion is formed by losing one or more electrons or gain one or

more electrons from their highest occupied energy levels.

There are two forms of ion, cation and anion.

CATION Cation is a positive ion.

Cations are formed by losing electrons in the highest

occupied energy levels.

By losing electrons, the number of protons and electrons are

not balanced, there are more protons than electron so the

atom became positively charged.

The charge for the cation is written as the charge followed

by a plus sign.

ANION Anion is a negative charged ion.

Anions are formed by gaining electron to make the highest

occupied energy level full.

By gaining electrons, there are more numbers of electrons than

protons in an atom so the atom became negative charged.

The charge for the anion is written as the charge followed by a

minus sign.

Practice problems Magnesium have the atomic number of 12, if it forms an ion, is it

going to form a cation or an anion?

How many electrons will it gain/lose?

(Magnesium have two valence electron on its outer energy level,

so it will become a cation by losing two electrons).

Ions are atoms which have

gained or lost one or more

electrons giving the ion a net

positive or negative charge.

Cation positively charged ion, it moves toward the

cathode (-).

Anion negatively charged ion, it moves toward the anode

(+).

Amphoteric substance can have a positive or negative or

zero charge, it depends on conditions.

Proteins are amphoteric compounds as they contain both

acidic and basic residues.

Each protein has ionizable groups ,amino or carboxyl

groups, in solution they give:

COOH + HOH COO- + H3O

NH2 + HOH NH3+ + OH

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.

At one pH, called the isoelectric point , 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’.

At this point the protein will not migrate in an electrical field

and remains at application point.

Every amino acid has its own specific isoelectric point.

When charged molecules are placed in an electric field,

they migrate toward either the positive (anode) or negative

(cathode) pole according to their charge.

Electrophoresis is the process of moving charged

biomolecules in solution by applying an electrical field across

the mixture.

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.

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.

Nucleic acids: DNA, RNA.

Factors influenced electrophoresis mobility:

The electric field.

Net charge of the molecule.

Size and shape.

Instrumentation and reagents: Two buffer boxes contain the buffer used in the process.

Each buffer box contains an electrode made of either

platinum or carbon, the polarity of which is determined by the

mode of connection to the power supply.

The electrophoresis support on which separation takes place

may contact the buffer directly, or by means of wicks.

The entire apparatus is covered to minimize evaporation and

protect the system.

The power supply to provide electrical power.

In most electrophoresis units, the gel is mounted between two buffer

chambers containing separate electrodes so that the only electrical

connection between the two chambers is through the gel.

General operations performed in conventional electrophoresis include:

(1) separation

(2) staining

(3) detection

(4) Quantification

Buffers

Function of buffer:

1. Carries the applied current.

2. Established the ph.

At one pH, called the isoelectric point , 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’.

At this point the protein will not migrate in an electrical field

and remains at application point.

Every amino acid has its own specific isoelectric point.

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

negative charge and will migrate towards the anode.

The speed and direction a charged particle moves is determined by:

Factors related to particle:

Size and shape.

Net charge (determined by buffer pH).

External factors :

Voltage.

Buffer pH.

Support medium (type of gel).

Temperature:↑speed but ↑↑ leads to denaturation of protein.

Electrophoresis of plasma protein

The proteins found in plasma 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

area in an electrical field.

o Normal total plasma protein concentration: 6 – 8.5 gm / dl.

o Electrophoresis separates serum Proteins into 5 distinct zones or

bands at barbital buffer at pH 8.6 :

Albumin.

Alpha globulin ( Alpha 1 and Alpha 2 ).

Beta globulin ( Beta 1 and Beta 2 ).

Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive protein.

The width of each band is dependent upon the number of

proteins that are present in that fraction.

Electrophoresis separates TSP into 5 distinct zones or bands

at ph 8.6:

Albumin

Alpha-1 globulin.

Alpha-2 globulin.

Beta globulin.

Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive

protein.

Q – WHAT is the difference

between serum proteins and

plasma proteins in electrophoresis ?

Serum:

is a clear yellowish fluid that remains from blood plasma after

clotting factors (fibrinogen, prothrombin ect.) that have been used in

the formation of a clot.

Plasma:

is a clear yellowish fluid that still contains all of the clotting factors

and have not been solidified into clot.

If plasma is used in electrophoresis , there will be an extra band

( fibrinogen ) between beta and gama bands.

Fibrinogen

Basic Procedure:

Sample is applied to an agarose gel.

Gel is placed into electrophoresis cell.

containing barbital buffer at pH 8.6.

Power is applied creating an electrical field and the proteins are

separated.

Proteins are fixed to the gel and stained.

Separated proteins on gel are scanned.

THANK YOU