Polyacrylamide gel electrophoresis (PAGE) allows researchers to separate proteins by size. Proteins are first denatured using heat and detergents to impart a uniform charge. They are then loaded into a discontinuous polyacrylamide gel system, where they are stacked and separated. The stacking gel prepares the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences through a process of electrophoresis using buffer systems. After separation, proteins can be visualized on the gel by staining. PAGE is a powerful technique that provides high resolution to analyze protein samples.

1. 1
Polyacrylamide gel electrophoresis (PAGE)
◘ properties of polyacrylamide gels :-
• Prepared by chemical copolymerization) ‫(بلمرة‬ of acrylamide monomers with a
crosslinking)‫(تشابك‬ reagent , usually N,N-methylenbisacrylamide .
• A clear Transparent gel is obtained , which is chemically inert )‫,(خامل‬
mechanically stable
• The reaction is a free radical polymerization , usually carried out with
ammonium persulfate as the initiator and ( TEMED ) as the catalyst .
The pore Size is exactly controlled with :-
• The total acrylamide concentration (T) (acrylamide monomers with a
crosslinking)‫(تشابك‬ reagent , usually N,N-methylenbisacrylamide )
• The degree of crosslinking (c), which is determined by the amount of cross-
linker (N,N-methylenbisacrylamide) relative to the total amount of acrylamide .
• The pore size decease with increasing ( T ) value .
• Investigators ( scientists ) are able to control the size of the pores in the gel by
adjusting the concentration of acrylamide , as well as the ratio of acrylamide to
bisacrylamide .
• Raising either the concentration of acrylamide or bisacrylamide , while holding
the other concentration constant , will decrease the pore size of the gel .
• Because Oxygen is a scavenger ) ‫(يلتهم‬ of free radicals , polymerization is
performed in closed cassettes . ) ‫لالكسجين‬ ‫يتعرض‬ ‫ال‬ ‫حتي‬ ‫مغلق‬ ‫وسط‬ ‫في‬ ‫البلمره‬ ‫تتم‬ (

2. 2
Chemistry of acrylamide polymerization
• Polymerization occurs because of free oxygen radicals that react with the vinyl groups
in acrylamide and bisacrylamide .
• The Oxygen radicals are generated from the catalyst , ammonium persulfate ( APS ) ,
when it reacts with a second catalyst ( TEMED ) .

3. 3
Proteins are denatured prior to electrophoresis
• Because proteins are so diverse with respect to either surface charges and geometries
• The molecular weight of folded proteins cannot be simply determined by their migration
rate in electric field .
• Positively and Negatively charged proteins would migrate in different directions !
• To resolve the proteins in a sample according to their size :-
- investigators must convert the proteins to uniform geometry and impart a uniform
(charge / mass ratio ) to the proteins .
‫ألن‬ ‫الموجب‬ ‫الكاثود‬ ‫إلي‬ ‫السالب‬ ‫األنود‬ ‫من‬ ‫يتجه‬ ‫عشان‬ ‫سالبه‬ ‫لشحنه‬ ‫هتكونا‬ ‫وهنا‬ ‫موحده‬ ‫شحنه‬ ‫ويكتسب‬ ‫يتكسر‬ ‫البروتين‬ ‫(الزم‬
‫هتروح‬ ‫واجزاء‬ ‫الموجب‬ ‫القطب‬ ‫ناحيه‬ ‫هتروح‬ ‫واجزاء‬ ‫تشتت‬ ‫يحصل‬ ‫وبكداه‬ ‫سالبه‬ ‫وشحنه‬ ‫موجبه‬ ‫شحنه‬ ‫ليه‬ ‫االساس‬ ‫في‬ ‫البروتين‬
) ‫السالب‬ ‫القطب‬ ‫ناحية‬
- In SDSPAGE , The solution is denature the proteins by boiling Them with the Anionic
detergent , Sodium dodecyl sulfate ( SDS) and 2-mercaptoethanol .

4. 4
• The combination of heat and detergent is sufficient to break
the many non-covalent bonds that stabilize proteins folds .
• and 2- mercaptoethanol breaks any covalent bonds
between cysteine residues .
• SDS consisting of a hydrophobic 12-carbon chain and
hydrophilic sulfate group .
• The SDS Hydrocarbon chain permeates ) ‫(يتخلل‬ the protein interior
and binds to hydrophobic groups , reducing the protein to a random
coil , coated with negatively charged detergent molecule all along its
length .
• Denatured proteins bind quite a lot of SDS , amounting to 1.4 g
SDS/g protein , or one SDS molecule for every two amino acids .

5. 5
Continuous and discontinuous buffer systems
Continuous buffer systems
• The identity and concentration of the buffer components are the
same in both the gel and the tank .
• Although continuous buffer system are easy to prepared and
give adequate resolution for some applications , bands tend to be
broader and resolution consequently poorer in these gels .
• These buffer systems are used for most forms of DNA ( eg :
Agarose gel electrophoresis )

6. 6
Discontinuous buffer systems
• Employ different buffers for tank and gel , and often two different
buffers within the gel .
• Discontinuous systems concentrate or “ stack “ the samples into
a very narrow zone prior to separation .
• Which results in improved band sharpness and resolution .
◘ The gel is divided into :-
- an upper (stacking) gel of low percentage of acrylamide and low
pH (6.8) and a (separation=running=resolving) gel with a pH of
8.8 and much smaller pores ( Higher percentage of acrylamide )
- The Stacking gel prevents any high-molecular-weight DNA
present in the sample from clogging )‫(انسداد‬ the pores at the top of
the running gel before low molecular-weight DNA has entered .
- both , the stacking and separating gels , contain only chloride as
the mobile anion , while the tank buffer contains glycine as its
anion , at a pH of 8.8 .

7. 7
The stacking and running gels underlie the resolving power of the SDS-PAGE gels
SDS-PAGE system can be considered a 3-component system :-
• The stacking and running ( resolving ) gels have different pore sizes , ionic strengths
and pHs .
• The third component is the electrophoresis buffer , which contains large amounts of
glycine .
• The Ionization state of glycine is critical to the separation .
• The sample is usually dissolved in glycine chloride buffer ( pH 8.9 ) before loading on
the gel .
• The glycine exists primarily in two forms
i.e Zwitter ions at low pH ( 6.9 – stacking gel ) and anion at high pH ( 8.9- resolving gel )

8. 8
Stacking Gel
• The sample pH is greater than the pH of stacking gel .
• After loading the sample on the well of the gel , the protein molecules present in the
sample are in anionic form .
• When electric fields is applied on the gel , the glycine-chloride buffer ions and sample
move in the stacking gel which has pH of 6.9 .
• in this pH , the glycine ion is in the form of zwitter ion with net charge zero and no
electrophoretic mobility .
• But the chloride ion and sample are in anionic form at pH 6.9 and act as mobile ions .
• The sample will tend to accumulated and form a thin , concentrated band sandwiched
between chloride and glycinate .
•The chloride ion and protein carry the most of the current .
The Resolving gel
• The ionic from of concentrated band reaches the resolving gel with pH 8.9 .
• The zwitter ionic glycine is changed into anionic form .
• In Resloving gel anionic glycine and chloride carry most of the current .
• The proteins present in the sample encounter with high pH and smaller pore size .

9. 9
The sample buffer
• contain a tracking dye , bromophenole blue ( BPB ) , which will migrate with the leading
edge of the proteins being separated on the gel .
• Also , contains glycerol , which allows the protein samples to settle into the bottom of
the gel wells .
• Once a voltage is applied , the chloride ions in the sample buffer and stacking gel move
rapidly toward the positive pole , forming the leading edge of a moving ion front .
Sodium Dodecyl Sulphate – Polyacrylamide ( SDS-PAGE)
• SDS binds to hydrophobic regions of denaturated protein chain in constant ratio of
about 1.4 g of SDS per gram of protein.
• The bound SDS molecules carrying negative charges mask the native charge of the
protein.
• The SDS binds to the unfolded proteins giving all proteins a similar shape ( i.e random
ciol or extend conformation ) and a uniform charge to mass ratio .

10. 10
◘ • ◘ A common way to detect proteins after electrophoresis is to stain the gel with
Coomassie blue , a gye that binds proteins .
- Gel are usually “ Fixed “ before staining with an acetic acid and methanol solution
which precipitates proteins into the acrylamide matrix .
Calculation of Relative mobility
• (RF) the distance the protein migrated is compared to the length of the gel or :-
Rf = Distance protein migrated / gel length

11. 11
PAGE
The lecture is Done ♥ ☺
Q.A