Western blotting in Molecular Biology and Biotechnology

 Blotting techniques involve transfer of biological samples (DNA,
RNA, or proteins) from a gel to a membrane followed by detection
on the surface of the membrane.
 Complexing the target with a labeled molecule for detection.
Blotting Techniques

Types of Blotting/Hybridization Techniques
1.Southern blotting
2. Northern blotting
3. Western blotting
4. Eastern blotting

Western blotting
 The Western blotting (alternatively, protein
immunoblotting)
 widely used analytical technique used to detect specific
proteins in the given sample of tissue homogenate or
extract.
 Method developed in the laboratory of George Stark at
Stanford. The name Western blot was given to the
technique by W. Neal Burnette in 1981.
 The SDS PAGE technique is a prerequisite for Western
blotting .

SDS-PAGE
 Most widely used method of analyzing protein mixture qualitatively.
 useful for monitoring protein purification.
 Based on separation of proteins according to size, can also be used to determine the
relative molecular mass of the proteins.
 PAGE separate proteins in fractions during electrophoresis process.
 Individual separated fractions can be examined.

Principle of Western blotting
 It uses gel electrophoresis where proteins are separated into
polyacrylamide gel according to their molecular weight.
 The proteins are then transferred to a membrane (typically
nitrocellulose or PVDF), where they are detected using antibodies
(both monoclonal and polyclonal antibodies) from mixture of
protein.

Steps in a Western blotting
1. Tissue preparation
2. Gel electrophoresis
3. Transfer
4. Blocking
5. Detection
6. Analysis

1. 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, homogenizer or by sonication.
 Western blotting is not restricted to cellular studies, bacteria, virus or
environmental samples can be the source of protein .
 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 and degradation.

2. Gel electrophoresis.
 The proteins of the sample are separated using gel
electrophoresis.
 Separation of proteins may be by isoelectric point
(pI), molecular weight, electric charge, or a
combination of these factors.
 By far the most common type of gel electrophoresis
employs SDS-PAGE.
 It is also possible to use a two-dimensional (2-D)
electrophoresis. Proteins are separated according to
isoelectric point in the first dimension, and according
to their molecular weight in the second dimension.

3. Transfer
 Proteins are moved from within the gel onto a membrane made
of nitrocellulose or polyvinylidene difluoride (PVDF).
 Transfer of proteins can be achieved either by capillary blotting
or by electroblotting.
 Capillary Blotting
1. The gel is placed on wet stack of buffer soaked filter papers
and membrane is placed on top of the gel.
2. Buffer is then drawn through the gel by placing a stack of dry
absorbent material followed by heavy weight on top of the
membrane.
3. The entire stack is placed in a buffer solution which moves up
through paper by capillary action, bringing the separated
proteins on to the membrane.
4. The process is carried out o/n.

 A quicker (a few hrs) and
more efficient method of
transfer.
 uses an electric current to
transfer proteins from the gel
into the PVDF or
nitrocellulose membrane.
 A sandwich of gel and
nitrocellulose membrane is
compressed in a cassette and
immersed in buffer.
 Current is passed, which
causes separated proteins to
electrophorese out of gel
onto the membrane.
 Nitrocellulose membrane
with transferred protein is
referred to as blot.
Electroblotting

 Protein binding is based upon hydrophobic interactions,
as well as charged interactions between the membrane
and protein.
 Nitrocellulose membranes are cheaper than PVDF, but
are far more fragile and do not stand up well to repeated
probing.

4. Blocking
 Step must be taken to prevent interactions between the membrane
and the antibody used for detection of the target protein.
 Blocking of non-specific binding is achieved by placing the blot in a
dilute solution of protein - typically 10% Bovine serum albumin
(BSA) or 5% non-fat dried milk in Tris-Buffered Saline (TBS), with a
minute percentage of detergent such as Tween 20 or Triton X-100.
 The protein in the dilute solution attaches to the membrane in all
places where the target proteins have not attached.
 This reduces "noise" in the final product of the Western blot, leading
to clearer results, and eliminates false positives.

5. Detection
Chemiluminescent or colorimetric detection: peroxidase

 The enzyme used in enzyme labelled Abs is usually either alkaline phosphatase which
converts 5 Bromo-4-chloro-indolylphosphate (BCIP) substrate into a blue product.
 or horseradish peroxidase with H2O2 as substrate, oxidizes 4-chloro-1 naphthol into
an insoluble blue product.
 an alternative approach to detect horseradish peroxidase is to use chemiluminescence.
 In the presence of H2O2 and luminol, HRP oxidizes luminol with emission of light.
 the light emission can be detected by exposing the blot to a photographic film.

Markers for Western blotting
 Enzymes are commonly used markers,
 125
I labelled secondary Ab.
 Flourescein isothiocyanate labelled secondary Ab.
 Gold labelled secondary Ab.
 Biotinylated secondary Ab.

Radioactive detection: X-rays
Radioactively labelled DNA can be used to detect DNA-binding proteins on
a blot.
 The blot is first incubated in a solution of radiolabeled DNA, then washed
and autoradiograph of the blot is made.
 The presence of radioactive bands detected on the autoradiograph,
identifies the presence of DNA binding proteins on the blot.

Western blotting can also identify a specific Ab in a mixture.
1. Known Ag of well defined molecular weight are separated by SDS-PAGE and
blotted onto the membrane.
2. Separated bands of known Ag are then probed with sample suspected of
containing Ab specific for Ag.
3. Rxn. of a Ab with a band is detected by using either radiolabeled or enzyme
linked secondary Ab specific for Abs in the test sample.

Applications of Western Blotting
 The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a
human serum sample.
 A Western blot is also used as the definitive test for Bovine spongiform encephalopathy
(BSE, commonly referred to as 'mad cow disease’).
 Some forms of Lyme disease testing employ Western blotting.
 Western blot can also be used as a confirmatory test for Hepatitis B infection.
 In veterinary medicine, Western blot is sometimes used to confirm FIV+ status in cats.

Western blotting in Molecular Biology and Biotechnology