Introduction
Ø SDS PAGE or Sodium Dodecyl
Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the
separation of proteins based on their molecular weight.
Ø It is a technique widely used in
forensics, genetics, biotechnology and molecular biology to separate the
protein molecules based on their electrophoretic mobility
Principle
of SDS-PAGE
Ø The principle of SDS-PAGE states
that a charged molecule migrates to the electrode with the opposite sign when
placed in an electric field.
Ø The separation of the charged
molecules depends upon the relative mobility of charged species.The smaller
molecules migrate faster due to less resistance during electrophoresis.
Ø The structure and the charge of the
proteins also influence the rate of migration.
Ø Sodium dodecyl sulphate and
polyacrylamide eliminate the influence of structure and charge of the proteins,
and the proteins are separated based on the length of the polypeptide chain.
Sample
preparation of SDS
Ø The protein sample is prepared by
heating them with SDS detergent and 2-mercaptoethanol until denaturation.
Ø SDS strongly binds to the protein
and forms a negative charge while 2-mercaptoethanol frees sulfhydryl groups
where polypeptide chains with an excess negative charge similar to mass ratio
get released.
Ø With the protein sample, the buffer
solution is added in microcentrifuge tubes and heat at 100°C for 3 minutes.
Ø Then the tubes are centrifuged at
15,000 rpm for 1 minute at 4°C
Sample preparation of page
Ø For preparing an electrophoretic
gel, several components including acrylamide, N, N’ –methylene-bis-acrylamide
(BIS), and a buffer solution is mixed together
Ø During polymerization of the gel,
ammonium persulfate, a free radical source, and a stabilizer is added and the
mixture is degassed or butanol is added to prevent bubble formation.
Ø A comb is inserted between the
spaces of the glass plate and allowed to polymerize.
Process
of SDS PAGE
Ø Boil the sample for 10 minutes to
completely denatures the proteins.
Ø Assemble the gel into the apparatus.
Ø Pour the buffer solution into the
chamber.
Ø Load 20ul the samples into the well.
Ø After that run electrophoresis by
connecting the current supplies.
Visualization
Ø The use of colored dyes is
non-specific i.e, it targets all protein and these stains are non-reversible as
well.
Ø This non-specific protein
visualization is used for quick quantification of samples in a gel.
Ø Specific protein visualization
requires the use of antibodies conjugated with a dye or enzyme that recognizes
the unique structure stern blotting is also associated with protein
visualization by moving proteins from a gel to a membrane that can be probed
with antibodies.
Ø The molecular mass of the unknown
protein sample is determined by comparing the distance traveled by the unknown
molecules with the marker.
Application
Ø It is used to measure the molecular
weight of the molecules.
Ø It is used to estimate the size of
the protein.
Ø Used in peptide mapping. It is used
to compare the polypeptide composition of different structures.
Ø It is used to estimate the purity of
the proteins.
Ø It is used in Western Blotting and
protein ubiquitination.
Ø It is used in HIV test to separate
the HIV proteins.