2. Principle
• PAGE (Polyacrylamide Gel Electrophoresis),
is an analytical method used to separate
components of a protein mixture based on
their size.
3. • PAGE is working upon the principle in which, the charged
molecule will migrate towards the appositive charged
electrode through highly cross linked matrix.
• Separation occurs due to different rates of migration
By the magnitude of charge and Frictional resistance
related to the size.
4. Structure of polyacrylamide:
• Electrophoresis using acrylamide gel is frequently referred
to as (PAGE) polyacrylamide gel electrophoresis.
• Polyacrylamide is a polymer of acrylamide and
N,N methylene bisacrylamide.
5. • Bisacrylamide is made up of two acrylamide molecules
linked by a methylene group, which is used as a cross
linking agent.
• Acrylamide monomers are polymerised in a head to tail
fashion into a long chain and occasionally , a
bisacrylamide molecule is introduced into the growing
chain, thus forming the second site for chain extension.
• In this way , a cross- linked matrix of a fairly well defined
structure is formed.
6.
7. • The polymerisation of acrylamide , an example of free
radicle catalysis, is initiated by the addition of :
• Ammonium persulphate and
• The base N,N,N,N-tetramethylenediamine (TEMED).
• TEMED initiates the decomposition of the persulphate ion
to give a free radicle.
9. Stacking gel
• It has pH of 6.8 units.
• Its poured over the separating gel after the polymerisation of
separating gel.
• Its used to stack proteins into a narrow band before they enter
into separating gel.
• Large pore size.
• Separation based on the difference between ionic strength and the
pH between the buffer and stacking gel
10. Separating gel
• It have a pH of 8.8 units
• Makes unto 85% of the gel body.
• Separation of protein based on their differential mobility.
• The concentration of separating gel usually varies from 7.5% to
15%
• Concentration mainly based on size of proteins under study.
• Small pore size compared to former.
11.
12. 1- Native-page:
2- Isoelectric Focusing-page
3- Denatured-page Or SDS-PAGE
4- Two Dimensional Gel Electrophoresis
TYPES OF PAGE
13. Native PAGE
• The molecules separate in an electric field on the basis of their net
charge and size of protein.
• Movement of molecules based on size.
• In this buffer is same in the upper and lower reservoir and in the
gel with the pH of 9.
• Bromophenol blue used as an indicator to check the progress of
electrophoresis.
• An electric current of ~300v is applied.
• Useful for separation and/or purification of mixture of proteins
14. Sds page
SDS page is a rapid, sensitive and widely used
technique from which one can determine the degree of
purity of a protein sample, the molecular mass of
unknown sample and the number of polypeptide
subunits with a protein.
15. • Smaller the molecule, the more easily it can migrate
through the gel.
• Hence the mobility increases with decrease in the
molecular weight.
• In most cases , SDS-polyacrylamide gel electrophoresis is
carried out with a discontinuous buffer system.
• Buffer in the reservoirs is of a different pH and ionic
strength from the buffer used to cast the gel.
16. • The sample and stacking gel contain Tris Hcl of pH 6.8
• Upper and lower buffer reservoirs contain Tris- glycine of pH
8.3
• The resolving gel contain Tris Hcl of pH 8.8.
• The ability of discontinuous buffer system to concentrate all of
the complexes in the sample into a very small volume greatly
increases the resolution of SDS-polyacrylamide gels.
• The effective range of separation depends on the concentration
of polyacrylamide and on the amount of cross linking.
17. • The size of these pores decreases with increase in
acrylamide:bisacrylamide ratio.
• Mostly of the ratio of 29:1.
20. 2-mercaptoethanol
• It’s a reducing agent.
• Breaks the intra and inter –chain disulphide bonds leaving
the denatured protein fully reduced.
• Separated into polypeptide chain.
21. Isoelectric focusing page
• In IEF, proteins are separated by electrophoresis in a pH gradient
in the gel
• They separate based on their relative content of positively or
negatively charged groups.
• Each protein migrate through the gel till it has no net charge i,e
isoelectric point.
• Polyacrlamide gel that has large pore size is used.
22. • Gel also contains mixture of polyampholates (small
multicharged polymer that have many pI values).
• With the application of E.F ,the polyampholate
migrate and produce a pH gradient.
• Each protein migrates through the gel until it reaches
a position at which pH is equal to pI.
• Isoelectric focusing can resolve proteins that differ in pI value by as
little as 0.01.
23.
24.
25.
26. Two dimensional gel electrophoresis:
• IEF can be combined with SDS –PAGE to obtain very high
resolution separations by a procedure known as Two dimensional
gel electrophoresis.
• In this technique protein sample is first subjected to IEF in a
narrow strip of gel containing polyampholytes.
• Then SDS-PAGE done by placing strip on top of SDS PAGE gel to
produce 2D pattern.
27. • Pattern of spots in which the protein have been
horizontally separated based on pI and vertically on their
mass.
• So proteins with similar pI get separated based on size and
vice versa
• 2D electrophoresis have enormous use in proteomics
studies
