ELECTROPHORESIS

history

In 1949, a team led by chemist Linus Pauling placed hemoglobin solutions from people with a disabling form of anemia and from healthy volunteers in an electric field, and found that the two samples migrated at different rates. In this way, the technique of electrophoresis helped decipher the molecular abnormality behind sickle cell disease

definition

Separation of NA and proteins according to size, based on movement through a gel medium when an electric field is applied.

Clinical aplications

Separation of serum protein , enzymes , hemoglobin's , lipids ,

Principle of electrophpresis

migration of charged molecules in solution in response to an electric field.

Cont…

Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving.

CLASSIFICATION

• Various types – defined by support used

• Paper – amino acids, small peptides

• Polyacrylamide – Proteins, small DNA/RNA (<500bp)

• Agarose – DNA/RNA

Polyacrylamide

is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%

is used in separation of Proteins, small DNA/RNA

Polyacrylamide is in liquid form at high temperature and gel for (semisolid) low or normal temperature Please check the gel getting hard by the addition of

bisacrylamide that forms cross linkage with the acrylamide

Polyacrylamide

Increase concentration of the gel will decrease pores → high selectivity

The way of spile the gel in the plate is vertical

Use spacer to separate the plates after adding the gel between the two

plates cool the gel Add the gel at power supply and connect

the cathode and the anode

Polyacrylamide

Remove spacer and add buffer Add the proteins (using the gel well)

Open the electric field Protein with different molecular weight

will be separated The movement of proteins will be to the

(+)

When I stop the process

during the movement of proteins you cant know when the protein reach the edge of gel

For that reason you should use marker molecule

Example for marker molecule (bromophenolblue)

how can I see the protiens

you should use protein stain Examples for protein stains Color Silver Stain 0.1 ng protein per band

Krypton Fluorescent Stain 2 ng protein per band

Imperial Stain (Coomassie R-250) 6 ng protein per band

GelCode Blue Stain (Coomassie R-250) 10 ng protein per band

Proteins

Proteins are amphoteric compounds In a solution with a pH above its

isoelectric point, a protein has a nett negative charge and migrates towards the anode

Below its isoelectric point, the protein is positively charged and migrates towards the

cathode. At a given pH therefore, and under non-

denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Uniforming the size of proteins denatures the proteins by heating I Stabilized the denaturation by SDS SDS is Sodium dodecyl sulphate is an anionic detergent which denatures

proteins by "wrapping around" the polypeptide backbone

and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1.

SDS confers a negative charge to the polypeptide in proportion to its length

SDS:Hydrophobic

Hydrophilic

FoldedproteinSDS

b-mercaptoethanolHeat

Charge ?

I can make all proteins with negative charge by adding

mercaptoethanol OR dithiothreitol

Intensity of the color is Proportional to

the Molecular weight Please check it

is the concentration

This is valid for agarose gel , so it is better to move this slide forward

Agarose

is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel

Used to separate DNA/RNA . Its horizintal Repaet the Polyacrylamide process

how can I see the N A

Dye Audioradiography 32P, Blotting

days…

ethidium bromide ( highly carcinogenic , binds DNA and fluoresces under illumination with UV light )

cyper green ( not carcinogenic , binds DNA and fluoresces under illumination with UV light )

both of them will not gives color until binding to DNA

Intinsity of the color proportional with molecular weight Be sure

Blotting

Blotting – Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization.

Hybridization – Process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix.

This slide and the next are not relevant and can be removed form the presentation

Cont…

Using specific probes that are labelled specific sequences of DNA can be identified.

There are three main hybridization techniques which vary in the sample blotted and the probes used;

1. Northern Blot-Transfer of an RNA sample separated and identified using DNA or RNA probes.

2. Southern Blot-Transfer of an DNA sample separated and identified using DNA or RNA probes.

3. Western Blot- Transfer of an Protein sample separated and identified typically using an antibody.

x

Ladder?

A ladder is a mixture of DNA fragments of selected sizes

When run in a gel electrophoresis, these fragments will separate into distinct bands that can be used as references

What is the Size of this Fragment?

Reference Ladder

An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band

This is OK , but previously U said that the intensity of the band is proportional to its molecular weight

Polyacrylamide

agarose

significantly more annoying to prepare than agarose gels

Agarose gels are extremely easy to prepare

Acrylamide is a potent neurotoxin It is also non-toxic

small range of separation, but very high resolving power

range of separation, but relatively low resolving power

For proteins For NA

NA remain negative at any pH used for

electrophoresis and in addition carry a fixed negative

charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid.

Electrophoretic separation of nucleic acids therefore is strictly according to size.

NA

Denaturing For DNA, RNA by urea

Other different type of electrophoresis

Two-dimensional electrophoresis

Insert here the 2D animation that I gave to U

Depend on isoelectric focusing and molecular weight of the proteins