Western blotting technique

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WESTERN
BLOTTING
BY KARMADIPSINH ZALA

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 The western blot (sometimes called the protein immunoblot)
is a widely used analytical techniqueused in molecular
biology, immunogenetics and other molecular biology disciplines
to detect specific proteins in a sample of tissue homogenate or
extract.

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 Synthetic or animal-derived antibodies are created that react
with a specific target protein.
 Next, the electrophoresis membrane is washed in a solution
containing the specific antibody.
 The excess antibody is then washed off, and a secondary
antibody that reacts with the first antibody is added.
 Through various methods such as staining,
immunofluorescence, and radioactivity, the secondary antibody
can then allow visualisation of the protein.

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 Other related techniques include dot blot analysis,
 quantitative dot blot,
 immunohistochemistry, and immunocytochemistry where
antibodies are used to detect proteins in tissues and cells
by immunostaining,
 and enzyme-linked immunosorbent assay (ELISA).

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Procedure
 Part – 1(protein isolation)
 Researchers uses chemical based / physical based Methods to
destruct the cell membrane and Release the proteins inside it
 Chemical based lysis uses various types of detergents to
perform cell lysis

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 Weak detergents are used to Extract water soluble proteins
without disrupting Intra cellular membrane compartmemts
 Strong detergents can solubilize or make it available of all
proteins found in cell

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• Supernatant is called protein lysate
which is used for western blotting

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• There are three type of lysates

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 Fourth commonly used lysate is immunoloprecipitate (IP)
 It is used to identify or detect protein of interest

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• Specific antibody
added to identify
protein of interest and
bind with it

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• Secondary antibody
bind with beads
.This complex used
to precipitate
protein antibody
complex

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• Cell lysis
disrupts the
carefully
controlled
cellular
environment
This can cause
the unwanted
protein
modification
to prevent this
lysis is
performed at
low

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Part - 2 (PAGE)

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(1) SDS - PAGE
 recipe for a traditional 10% Tris-glycine mini gel for SDS-PAGE:
 7.5 mL 40% acrylamide solution
 3.9 mL 1% bisacrylamide solution
 7.5 mL 1.5 M Tris-HCl, pH 8.7
 Add water to 30 mL
 0.3 mL 10% APS
 0.3 mL 10% SDS
 0.03 mL TEMED

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 SDS-PAGE is used for routine separation and analysis of
proteins because of its speed, simplicity, and resolving capability
 SDS-PAGE, the gel is cast in a buffer containing sodium dodecyl
sulfate (SDS), an anionic detergent. The protein samples are
heated with SDS before electrophoresis so that the charge
density of all proteins is made roughly equal.

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 The SDS (aided by heat) denatures proteins in the sample and
binds tightly to the uncoiled molecules. Usually, a reducing agent
such as dithiothreitol (DTT) is also added to cleave protein
disulfide bonds and ensure that no tertiary or quaternary protein
structure remains.
 Consequently, when these samples are electrophoresed,
proteins separate according to mass alone, with very little effect
from compositional differences.

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NATIVE PAGE
 In native PAGE, proteins are separated according to the net
charge, size, and shape of their native structure.

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 Electrophoretic migration occurs because most proteins carry a
net negative charge in alkaline running buffers.
 The higher the negative charge density (more charges per
molecule mass), the faster a protein will migrate. At the same
time, the frictional force of the gel matrix creates a sieving effect,
regulating the movement of proteins according to their size and
three-dimensional shape.
 Small proteins face only a small frictional force, while larger
proteins face a larger frictional force. Thus native PAGE
separates proteins based upon both their charge and mass

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 Because no denaturants are used in native PAGE, subunit
interactions within a multimeric protein are generally retained
and information can be gained about the quaternary structure.
 In addition, some proteins retain their enzymatic activity
(function) following separation by native PAGE. Thus, this
technique may be used for preparation of purified, active
proteins.

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 Following electrophoresis, proteins can be recovered from a
native gel by passive diffusion or electro-elution. To maintain the
integrity of proteins during electrophoresis, it is important to keep
the apparatus cool and minimize denaturation and proteolysis.
pH extremes should generally be avoided in native PAGE, as
they may lead to irreversible damage, such as denaturation or
aggregation, to proteins of interest.

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1d PAGE
 The most common form of protein gel electrophoresis is
comparative analysis of multiple samples by one-dimensional
(1D) electrophoresis. Gel sizes range from 2 x 3 cm (tiny) to 15 x
18 cm
 )

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 Protein samples are added to sample wells at the top of the gel.
When the electrical current is applied, the proteins move down
through the gel matrix, creating what are called "lanes" of protein
"bands". Samples that are loaded in adjacent wells and
electrophoresed together are easily compared to each other
after staining or other detection strategies.
 The intensity of staining and "thickness" of protein bands are
indicative of their relative abundance. The positions (height) of
bands within their respective lanes indicate their relative sizes
(and/or other factors affecting their rate of migration through the
gel).

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2D – PAGE
 Multiple components of a single sample can be resolved most
completely by two-dimensional electrophoresis (2D-PAGE).
 The first dimension separates proteins according to their native
isoelectric point (pI) using a form of electrophoresis called
isoelectric focusing (IEF). The second dimension separates by
mass using ordinary SDS-PAGE.

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 To perform IEF, a pH gradient is established in a tube or strip gel
using a specially formulated buffer system or ampholyte mixture.
Ready-made IEF strip gels (called immobilized pH gradient
strips or IPG strips) and required instruments are available from
certain manufacturers. During IEF, proteins migrate within the
strip to become focused at the pH points at which their net
charges are zero. These are their respective isoelectric points.
 The IEF strip is then laid sideways across the top of an ordinary
1D gel, allowing the proteins to be separated in the second
dimension according to size.

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Part – 3
(Transfer,blotting and visualization)
Proteins are transferred from gel to membrane
TO

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Western blotting is an electrical illusion
where electric field is applied to facilitate
the Migration of the protein

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Gel and membrane are placed in buffer
chamber where the negative Charge is
close to Gel and positive charge is close
to membrane

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 Blocking agent is added to membrane for minimize the non-
specific binding of antibody
 After that , membrane is incubated in solution containing
blocking Solution and Primary antibody that specifically
recognizes the protein of interest.
 Membrane is washed in washing Buffer in series.
 Which contains blocking Solution to remove non specifically
bound primary antibodies

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 Secondary antibody is added which recognizes used primary
antibody and bind to it .
 Secondary antibody Is conjugated to enzyme which enjoyed
light i.e.Horseradish peroxidase.
 After that , the x ray film is placed on the Membrane

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