Electrophoresis

                          ELECTROPHORESIS- I                                     

What is Electrophoretic ?

It is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

It is used in 
1. clinical chemistry to separate proteins by charge and/or size. 
2. Biochemistry and Molecular biology to separate DNA and RNA fragments by length, or to separate proteins by charge.

                                                            Fig.- Electrophoresis machine

GENERAL PRINCIPLES

❑The term electrophoresis describes the migration of a charged particle under the  influence of an electric field.
❑Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionizable.
❑ A. Tiselius: Nobel Prize for Chemistry in 1948.

What does it do?

Separation of
 • Proteins 
* Western Blots
* SDS-PAGE
• Nucleic Acids
* Northern Blots
* Southern Blots

• Based on 
* Charge and/or
* Size

Parts of the System

• Gel Support Medium 

* Agarose 

* Polyacrylamide (PA) 

* Native Gels 

# Use PA or Starch 

# No Denaturant 

• Buffer

• DC Power Supply

Electrophoresis units are available for running either 

1. vertical or
2. horizontal gel systems.

                                                            Fig.- Vertical electrophoresis

                                                       Fig.- Horizontal Electrophoresis

SUPPORT MEDIA

1. Agarose gels 
2. Poly acrylamide

The pioneering work on protein electrophoresis by Arne Tiselius (for which he received the Nobel Prize in Chemistry in 1948) was performed in free solution.

Agarose gel 

❖ Agarose is a linear polysaccharide (average relative molecular mass about 12 000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose.

❖ isolated from certain seaweeds

❖ inter- and intramolecular hydrogen bonding within and between the long agarose chains. 

❖ Agarose gels are used for the electrophoresis of both proteins and nucleic acids.

Preparation of agarose gel 

• Agarose sets thermally. 

• Agarose gels are extremely easy to prepare you simply mix  agarose powder with buffer solution, melt it by heating, and  pour the gel. 

• It is also non-toxic except after adding of ethidium bromide.

Safer alternatives to Ethidium Bromide

• Methylene Blue 

• BioRAD - Bio-Safe DNA Stain 

• Ward’s - QUIKView DNA Stain 

• Carolina BLU Stain

                                                        Fig.- Preparation of Agarose gel

advantages                                                           disadvantages

Inexpensive                                                          Less sensitive

Less toxic                                                              More DNA needed on gel

No UV light required                                      Longer staining/destaining time

No hazardous waste disposal

Continue......  Electrophoresis -II