1. Electrophoresis
PAGE
Dr Gihan Gawish

2. Types of Gels The most common types of gels are:
Starch gels: seldom used nowadays
Agarose gels: for separation of nucleic acids and large proteins
Polyacrylamide gels: for separation of most proteins and small nucleic acids
Dr Gihan Gawish

3. Polyacrylamide Gel Electrophoresis (PAGE)
PAGE can be classified according the separation conditions into:
1- Native-PAGE:
Separation is based upon charge, size, and shape of macromolecules.
Useful for separation and/or purification of mixture of proteins
This was the original mode of electrophoresis (introduced in 1930s, Nobel Prize 1948).
2- Denatured-PAGE or SDS-PAGE
Separation is based upon the molecular weight of proteins.
The most common method for determining MW of proteins
Very useful for checking purity of protein samples
Dr Gihan Gawish

4. PAGE 3- Isoelectric Focusing-PAGE
Separation of basis of pI, not MW
Recently, became popular as a part of proteomic techniques
PAGE can also be classified according to the physical shape of the gel:
slab (most common) or tubes
Continuous, discontinuous, stacked, or gradient gel
One dimensional or two-dimensional electrophoresis
Dr Gihan Gawish

5. SDS-PAGE
Biopolymers such as proteins and nucleic acids are folded into compact structures
They held together by a variety of non- covalent, ionic interactions such as
hydrogen bonding
salt bridges.
Dr Gihan Gawish

6. SDS-PAGE
The electrophoretic mobility of the denaturated molecule will be changed, compared to that in non denaturating conditions
It migrates as an unstructured monomer through the electrical field.
Dr Gihan Gawish

7. SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SDS-PAGE, is a technique widely used to separate proteins according to their electrophoretic mobility
The SDS gel electrophoresis of samples having: identical charge to mass ratios results in fractionation by size
Dr Gihan Gawish

8. SDS-PAGE disrupting its compact folded structure. Dr Gihan Gawish
The detergent sodium dodecyl sulphate (SDS) consists of:
hydrophobic 12-carbon chain
polar sulphated head
Hydrophobic chain can Intercalate into Hydrophobic parts of the protein by detergent action
disrupting its compact folded structure.
Dr Gihan Gawish

9. Dr Gihan Gawish

10. SDS-PAGE
SDS is highly charged (-)
binds to proteins in proportion to their molecular weight
SDS molecules exert strong internal repulsive forces
tend to stretch out the random coil protein into a rod-like shape
All proteins are made to look virtually the same rod diameter but with lengths that are proportional to the molecular weight of the protein.
Dr Gihan Gawish

11. SDS-PAGE: Procedure
The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non– disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass
Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure.
This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
Dr Gihan Gawish

12. SDS page
Dr Gihan Gawish

13. SDS PAGE in practice
Denatured SDS-protein mixture with a colored dye or stain added for tracking is loaded at the top of a slab or tube of a gel (typically polyacrylamide)
Electric field imposed within the gel using electrodes attached to a power supply
Proteins and dye migrate down the gel at a constant rate that depends on the molecular weight of the protein
Smaller proteins migrating faster
Dr Gihan Gawish

14. SDS PAGE in practice
Over a limited molecular weight range, the electrophoretic mobility of proteins is found to be proportional to the logarithm of their molecular weight
Dr Gihan Gawish

15. SDS- PAGE Advantages
Rapidly and cheaply measure molecular weights with an accuracy of about 5%
determine trace amounts of impurities in a sample
Dr Gihan Gawish

16. Preparative SDS-PAGE
Samples are electrophoresed (native or SDS PAGE) through a cylindrical gel
Bands pass through a thin frit within the elution chamber
Isolated bands are drawn radially by a pump onto a fraction collector into discreet liquid fractions
Dr Gihan Gawish

17. Dr Gihan Gawish

18. Isoelectric focusing-PAGE
Isoelectric focusing is a technique for separating different molecules by their electric charge differences.
The charge of molecule changes with the pH of its surroundings.
A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the negative electrolode.
Dr Gihan Gawish

19. Isoelectric Focusing (IEF)-PAGE
Employs a pH gradient extending the entire gel: (slabs or tubes)
Carrier ampholytes are used to set up the pH gradient
Protein sample is applied:
no SDS. charges make proteins different
Dr Gihan Gawish

20. IEF-PAGE At pH = pI, a protein will have no net charge  stop moving
At any other pH in the gradient, the protein has either a positive charge (pH<pI) or negative charge (pH>pI)
Runs requires higher voltages and longer periods of time, but gives resolution up ±0.001 pH
Dr Gihan Gawish