i am HAFIZ M WASEEM from mailsi vehari BSc in science college Multan Pakistan MSC university of education Lahore Pakistan I love Pakistan and my teachers

2. HAFIZ
MUHAMMAD
WASEEM

3. • Contents:
• Electrophoresis
• Types of Electrophoresis
• Acrylamide
• Polyacrylamide
• Gel Electrophoresis Technique
• Polyacrylamide Gel Electrophoresis
• Principles of PAGE (SDS-PAGE protocol)
1. Sample preparation
2. Gel preparation
3. Electrophoresis(passage of voltage across gel)
4. Staining and visualization
• Conclusion
POLYACRYLAMIDE GEL ELECTROPHORESIS(PAGE):

4. ELECTROPHORESIS :
• Electrophoresis is a technique which separates the macromolecules on the basis of
their size and their movement in the electric field on the basis of their size.
• This technique is being widely used in biotechnology and macrobiology.
• Larger and smaller molecules are separated in this technique from their constituents
and then they are studied and further categorized accordingly.
• Even these molecules which are separated have exposed a huge experimental
panorama for the biologists and other science dealers. The best example for this is
the cloning technique and the hybridization for the increase in productivity.

5. TYPES OF GELS PERFORMING ELECTROPHORESIS:
• There are mainly two types of gels performing electrophoretic technique:
• Agarose gel electrophoresis and Polyacrylamide gel electrophoresis. But
somewhere starch is also being used.
1. PAGE is used for the separation of big molecules such as proteins etc.
which ranges in size from 5 to 2000 kDa. Gel prepared for it’s separtion is
of low concentration so that the holes of gel are large enough for the
passage of these big molecules.
2. Agarose gel electrophoresis is for the small and smallest fragments under
study such as nucleic acids mainly including DNA. It’s gel concentration is
maximum so that the pore size should be small for their passage. Acids
range from 5 to 200 kDa.

7. ACRYLAMIDE
• Acrylamide is an organic compound with chemical
formula CH2=CHC(O)NH2.
• It is a white , odorless solid which is soluble in water
and many organic solvents. Acrylamide is found in some
foods. It can be produced when vegetables that contain
the amino acid asparagine, such as potatoes , are
heated to high temperatures.
• It is also a component of tobacco smoke.

8. POLYACRYLAMIDE GELS:
• Polyacrylamide gels are the polymers of
acrylamide so these are formed by the
polymerization of acrylamide in the presence of
a cross linking reagent which is commonly
named N,N'-methylenebisacrylamide via a free
radical initiated vinyl polymerization
mechanism.
• Ammonium persulphate is usually used as the
free radical initiator while N,N,N’,N’-
tetramethylenediamine (TEMED) stabilizes the
polymerization reaction.

9. GEL ELECTROPHORESIS TECHNIQUE:
• Gel electrophoresis is a technique which is being
used in the biochemistry, genetics, molecular
biology, forensic chemistry and biotechnology in
which the macromolecules, usually proteins or
nucleic acids, are separated on the basis of
electrophoretic mobility.
• Electrophoretic mobility is the function of length
conformation and charge of the molecule.

10. POLYACRYLAMIDE GEL ELECTROPHORESIS:
• Polyacrylamide gels are also used for the
electrophoretic functions in which the
respective protein or nucleic acids are placed
and they move across an electric field and
moves toward the oppositely charged
electrode.
• The rate of their movement in electrophoretic
system is governed by various factors such as
temperature , pH, and buffer conditions in
addition to intrinsic properties such as size,
shape and charge of molecules.

11. POLYACRYLAMIDE GEL ELECTROPHORESIS:
• Electrophoretic separation strictly on the basis of their molecular weight is
possible only if the charge of the molecules is manipulated to the same sign.
In such a case the mobility of protein molecules will be solely raliant on their
size.
• PAGE is a technique based on this idea and separates the protein molecules
on the basis of their size.

12. PRINCIPLES OF PAGE:
• In PAGE an anionic detergent called sodium dodecyl sulphate(SDS) binds with orotein
molecules giving them the negative sign. Now, all the protein molecules move across the
electric field over the gel.
• Loose gels(4-8%acrylamide) aloow higher molecular weight molecules to move faster
through the gel while the hard gels(12-20% acrylamide) restrict the migration of large sized
molecules and allow only small sized molecules to pass through the gel. That’s why hard
gels are avoided for the technique.

13. SDS-PAGE PROTOCOL:
1- SAMPLE PREPARATION
• Protein samples are denatured by heating them with a detergent SDS and
mercaptoethanol. The former(SDS) binds strongly to the proteins and gives
them a high negative charge whilst the latter (mercaptoethanol) frees
sulfhydryl groups, thus yielding polypeptide chains carrying an axcess negative
charge and similar charge to mass ratio.
• This helps the resolution of proteins strictly based on their size during gel
electrophoresis.

14. 2-GEL PREPARATION:
• The components of electrophoretic gel includes acrylamide, BIS, and a buffer.
• The mixture is degassed to prevent bubble formation during polymerization of the gel.
• Ammonium persulphate, a free radical source and a stabilizer are added to start
polymerization. BIS is also added to form cross-links between acrylamide molecules until a
gel is ultimately formed.

15. 3-ELECTROPHORESIS:
• As the electric current is applied through the
gel, the proteins molecules start moving
towards the positive electrode as they are
oppositely charged.
• Each molecule moves at different rate
depending upon their molecular weight.
Small molecules move rapidly and high
voltage also affects the movement at faster
speed. After a few hours, the protein
molecules are all separated by size.

16. 4-STAINING AND VISUALIZATION:
• Once, electrophoresis is complete ; the gel can be stained using coloured dyes
such as Coomassie Brilliant Blue or ethidium bromide to make the separated
proteins appear as distinct coloured bands on the gels. Excessive dye is
removed after staining and the coloured intensity is measured of the dried gel
.
• Some gel systems introduce a tracking dye such as bromophenol blue along
with protein samples. The visible distance travelled by the dye on the gel
helps in deciding the required duration of electrophoresis.
• Electrophoresis needs to stop at a certain point so that no protein molecule
leave the gel and gets into the buffer.

18. CONCLUSION:
• Proteins and nucleic acids are first denatured and then their charged are manipulated as
a same charge. After this charge conversion they are placed in the respective gel
experiencing an electric voltage. This moves the molecules according to their size and
appears on the gel.

19. REFERENCES
1. https:/www.bio.davidson.edu/courses/genomics/methods/sdspage.html#P
AGE
2. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_
6040.pdf
3. https://www.udel.edu/biology/fschmieg/411acrylamide.htm.
4. Socratic.org/question.com.
5. https://www.news.medical.net.com