Western blot or western blotting Tyes of western blotting Southern blot, Northern blot and western blot

2. Westernblot
A PRESENTATION BY
AYAZ KHAN,MARYAM ALTAF,HOORULIN

3. WESTERN BLOT

4. Topics include..
Introduction to WESTERN BLOT…
DIFFERENT STEPS INVOLVED IN WESTERN BLOTTING
o Tissue preparation
o Gel electrophoresis
o Transferring
o Blocking
o Detection
Analysis
Applications
LIMITIATIONS OF WESTERN BLOT

8. What is BLOTTING
Blots are techniques for transferring DNA, RNA
and proteins onto a carrier so they can be
separated and often follows the use of Gel
Electrophoresis.The southern blot is used for
transferring DNA while the Northern blot is for
RNA and the western blot is for Protein

9. Types of blotting techniques
Blotting Techniques
Southern blottin used to
detect DNA
Northern blotting
Used to detect RNA
Western blotting
used to detect
proteins

10. Western Blotting
DEFINITION..
Western blotting is a widely used analytical
technique in molecular biology to detect specific
protein in a sample of tissue homogenate or extract.
It works on the principle of gel
electrophoresis.Protiens are separated based on
their size on polyacrylamide gel

11. Principles 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 pre requisite for western
blotting

12. Steps
Tissue preparation
Gel electrophoresis
Transfer
Blocking
Detection

13. Tissue preparation
Samples can be taken from whole tissues
,cell.culture,bacteria, viruses environmental samples
etc that are homogenized in a buffer to protect the
protien of interest in from degradation
 ......solid tissues broken down mechanically (e.g. by blender).
Detergents, salts or buffer may be added to
encourage lysis( breaking of cell membranes ) and
solubilize proteins..
Done at low temperature to prevent protein
denaturation .

14. Gel electrophoresis
In gel electrophoresis we separate the proteins on the basis of
o Isoelectric point
o .Molecular weight.
o Electric charge.
PROCEDURE
 Most common type of gel used are
 Polyacylamide gels and buffers loaded with sodium dodecyl sulfate(SDS).
 SDS-PAGE.
 Concentration of acylamide.
 Sample are loaded in wells.
 Voltage is applied.
 Usage of two-dimensional gels

16. Transfer
Proteins moved from within the gel to a membrane made of
nitrocellulose or polyvinylidene difluoride (PVDF)
Membrane is placed on top of gel, with a stack of filter papers placed
on top of that
Entire stack is placed in a buffer solution, which moves up the paper
through capillary action, bringing the proteins up with it-
proteins are exposed on a thin surface layer for detection
Another method is called electro blotting, where an electric current
pulls the proteins from the gel to the membrane

17. Blocking
As the membrane is able to bind protein, steps are taken to prevent
interactions between the membrane and the antibody used for
detecting the target protein.
◦ in small letter..- membrane placed in in a dilute solution of protein with a
minute percentage of detergent.
◦ protein in the dilute solution attaches to the membrane in all the places
where the target protein has not.
◦ no room,, for added antibody to bind onto the membrane other than
binding sites of the target protein- this ensures clearer results and eliminates
false positives

19. Detection
Membrane Is "probed" for protein of interest wit a modified antibody
 Antibody linked to a reporter enzyme
 Drives a colorimetric reaction and produces a colou when exposed to an
appropriate substrate
This takes place in 2-step process:
 A primary antibody is added at an appropriate dilution and incubated with
the membrane. It will bind to the target protein if it is present
 Membrane rinsed to remove unbound primary antibody. In order to detect
the antibodies which have bound, a second antibody (or 'conjugate") is
added. These are ant immunoglobulin antibodies.

20. Detection continue
oAfter excess second antibody is washed off, a substrate is added-
o precipitates upon reaction with the conjugate resulting in a visible band
where the primary antibody bound to the protein
oAn isotope-labeled primary antibody can also be used, which can be
detected directly by X-ray film and does not require the secondary
antibody

22. Applications
Some common applicatios of western blotting are
Medical diagnosis
Hiv test through human serium sample
Bovine spongiform encephalopathy
 lypme disease
 hepatitis B

23. Analysis
It is important to understand the advantages and disadvantages of
different ways of detecting a protein of interest on the membrane.
Here, we present the TWO main types of protein detection for western
blot
Radioactive detection
Flourescent detection

24. Radioactive Detection
Radioactive detection is One of the first methods of primary antibody
detection to be developed was the use of secondary antibodies labeled
with radioisotopes (PMID: 388439). Iodine-125 was a popular choice
with a relatively long half-life and emission of gamma rays consisting of
low-energy photons that can be detected easily using X-ray films. Once
a popular technique, its use nowadays is relatively rare

25. Advantages and disadvantages of
radioactive detection
Advantages:
◦ Radioactive detection offers good sensitivity and is very quantifiable.
Disadvantages :
• Quite expensive.
• Dangerous – as radio active rays are used.

26. Flourescent Detection
In this method, the detection antibodies are not conjugated
with enzymes but with fluorophores. Fluorophores are excited
by light of a specific energy, which leads to the emission of
light at a longer wavelength (e.g.; 680 nm for excitation and
694 nm for emission)
Advantages
oMultiplexing
oStability
oSimple to use
Disadvantages
o Requires detection equipment
o Sensivity

27. Limitations of western blotting
Limitations of Western Blotting Very delicate and time
consuming process. A minute imbalance at any level of the
procedure can skew the results of entire process
Cause erroneous in bands or no bands due to insufficient
transfer..
 Well trained techniques are required for this technique.
Primary antibody availability is crucial
It is just a Semi-Quantitative test. Only estimation and not a
precise .
measurement of molecular weight of the protein is possible.

28. Question session