Western blotting

2. PRESENTED BY
Raja Muhammad Munsif
Mudasar Majeed
Raja Qadir Iqbal
Khawaja Waqas Ahmed
Shahzad Naseer Awan
UNIVERSITY OF AZAD JAMMU AND KASHMIR MUZAFFARABAD
DEPARTMENT OF BOTANY

3. WESTERN BLOTTING
Defination
 The Western Blot is an analytical technique used to detect
specific proteins in a given sample of tissue homogenate or
extract.
 Western blotting is an immunoblotting(protein detection)
technique used for the separation, to assess the presence,
amount and molecular- weight of proteins in cellular or tissue
extracts by using antibodies.

4. Western Blotting – Used to
detect protiens
Southern Blotting - Used to
detect DNA
Northern Blotting - Used to
detect RNA
TYPES OF BLOTTING
TECHNOQUES

5. PRINCIPLE OF WESTERN
BLOTTING
 Western blotting is an Immunoblotting technique which
rely on the specificity of binding between a molecule of
interest and a probe to allow detection of the molecule of
interest in a mixture of many other similar molecules.
 In Western blotting, the molecule of interest is a protein
and the probe is typically an antibody raised against that
particular protein.
 The SDS PAGE technique is a prerequisite for Western
blotting

6. THE PROCEDURE INCLUDES
 Tissue preparation
 Gel electrophoresis
 Transfer
 Blocking
 Detection
 Analysis

7. TISSUE PREPARATION
 Samples may be taken from whole tissue or from
cell culture.
In most cases, solid tissues are first broken down
mechanically using a blender.
 It should be noted that bacteria, virus or
environmental
samples can be the source of protein and thus
Western
blotting is not restricted to cellular studies only.
 Assorted detergents, salts, and buffers may be
employed to
encourage lysis of cells and to solubilize proteins.
 Tissue preparation is often done at cold
temperatures to
avoid protein denaturing.

8. GEL ELECTROPHORESIS
 The proteins of the sample are separated
using gel
electrophoresis. Separation of proteins may be
by
isoelectric point molecular weight, electric
charge, or a
combination of these factors.
 The principle involved is the difference in the
ELECTROPHORETIC MOBILITIES of different
proteins

9. SDS-PAGE
 The SDS-PAGE is sodium dodesylsulphate
polyacrylamide gel electrophoresis
 SDS-PAGE is widely used in biochemistry, forensics,
genetics and molecular biology to separate the
proteins according to their electrophoretic mobility
 Discontinuous polyacrylamide gel is commonly used
as supporting medium in separating proteins by
electrophoresis and sds (lauryl sulphate) to denature
the proteins.

10. APPARATUS OF SDS-PAGE
 Casting Stand
 Clamping Frame
 Sample loading guide
 Glass plates
 Plastic comb
 Casting frame

11. PRINCIPLE OF SDS-PAGE
Proteins move in
the electric field.
Their relative
speed depends
on the charge,
size, and shape of
the protein

12. TRANSFERING
Transfer step
The transfer of the proteins
onto the nitrocellulose
membrane.
The proteins separated on the
SDS-PAGE gel are trasferred to
the membrane by using
electrophoresis. The
localization of the proteins do
not change.

13. TRANSFER APPARATUS
Two major types
Semi-dry apparatus
No transfer buffer chamber
Only has plates (runs for hours)
Submerge transfer apparatus
Contains a chamber filled with
transfer buffer
Electrodes
-Plate electrodes (runs for hours)
-Wire electrodes (runs overnight)

14. TRANSFER THE PROTEIN FROM THE
GEL TO THE MEMBRANE
Transfer of the proteins
fractionated by SDS-PAGE to a
solid support membrane
(Western blotting) can be
accomplished by electro
blotting

15. RUNNING A TRANSFER
Similar to running a gel
Negatively charged proteins run
towards the anode end of the
transfer apparatus
Always have nitrocellulose on
the anode side to capture
proteins.

16. PVDF MEMBRANE
We can also use another membrane like
PVDF membrane
Why use it?
-Stronger than nitrocellulose and able to strip
phosphate groups off proteins.
-Must soak in methanol first since it doesn’t
become wet in water

17. AFTER TRANSFER
Membrane is washed in a Tris buffer
saline Tween 20 solution (TBST).
Block membrane in non-fat dried milk
solution. Usually 5% w/v. Prevents
unwanted binding of antibodies to
membrane for 1 hr
Washing with TBST to remove blocking
solution

18. BLOKING
 The membrane has the ability to bind to proteins in this case
both the target and antibodies are proteins and so there
could be some unwanted binding.
 Blocking of non-specific binding is achieved by placing the
membrane in a dilute solution of protein - typically Bovine
serum albumin(BSA) with a minute percentage of detergent
such as Tween 20.
 The protein in the dilute solution attaches to the membrane
in all places where the target proteins have not attached.
Thus, when the antibody is added, there is no room on the
membrane for it to attach other than on the binding sites of
the specific target protein.

19. THIRD STEP
Primary antibody
incubation step
The primary antibodies
which specifically
recognize the proteins
of interest are used.

20. PRIMARY ANTIBODIES
Proteins that bind to specific
epitopes on specific
proteins.
Two main types used in
western blotting
Monoclonal
Polyclonal

21. ANTIBODIES FOR WESTERN
BLOTTING
Monoclonals are to one epitope
while polyclonals are to many
epitopes on the protein of
interest. If epitope changes upon
denaturing of the protein it will
not recognize the protein

22. FOURTH STEP
Secondary antibody incubation step.
Use of secondary antibody which recognizes the primary antibody used
in the third step.
Wash at least three times with TBST for 5-10 minutes each
Added antibodies against the animal that the primary antibodies were
made in.
These antibodies are also conjugated with enzymes such as peroxidase
The secondary antibodies are added to the membrane and incubated at
room temperature for one hour.

23. FIFTH STEP
Visualization step
Making the antigen-antibody complex
visible (staining)
Autoradiography (radioactive P /
chemiluminescence)
Avidin-biotin complex
Fluoresence method.

24. ANALYSIS
 After the unbound probes are washed away, the
western
blot is ready for detection of the probes that are
labeled
and bound to the protein of interest.
 Size approximations are taken by comparing the
stained
bands to that of the marker loaded during
electrophoresis.
 The process is repeated for a structural protein,
such as
actin or tubulin that should not change between
samples.

25. ADVANTAGES
 While ELISA being a non specific test, Western blotting is a
more specific test for detection of HIV.
 It can detect one protein in a mixture of proteins while giving
information about the size of the protein and so is more specific.
This method is, however, dependent on the use of a high-quality antibody directed against
a desired protein.
 This antibody is used as a probe to detect the protein of interest.
 Detects proteins and estimates their molecular weight.
 Detects changes in phosphorylation and lipid- modifications.
 Used to detect changes in protein expression.