Dr Gihan Gawish

1

PAGE

Types of Gels

Dr Gihan Gawish

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

2

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

PAGE

Dr Gihan Gawish

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

4

Dr Gihan Gawish

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.

5

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

6

SDS-PAGE

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis

Dr Gihan Gawish

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

7

SDS-PAGE

Dr Gihan Gawish

8

Dr Gihan Gawish

9

SDS-PAGE

Dr Gihan Gawish

All proteins are made to look virtually the same rod diameter but with lengths that are

proportional to the molecular weight of the protein.

10

SDS-PAGE: Procedure

Dr Gihan Gawish

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.

11

Dr Gihan Gawish

12

SDS PAGE in practice

Dr Gihan Gawish

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

13

SDS PAGE in practice

Dr Gihan Gawish

Over a limited molecular

weight range, the

electrophoretic mobility of

proteins is found to be

proportional to the logarithm

of their molecular weight

14

SDS- PAGE Advantages

Dr Gihan Gawish

Rapidly and cheaply measure molecular weights with an accuracy of about 5%

determine trace amounts of impurities in a sample

15

Preparative SDS-PAGE

Dr Gihan Gawish

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

16

Dr Gihan Gawish

17

Isoelectric focusing-PAGE

Dr Gihan Gawish

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.

18

Dr Gihan Gawish

19

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

Isoelectric Focusing (IEF)-PAGE

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

20