This document discusses different blotting techniques used to detect proteins, DNA, and RNA. It focuses on the Western blot technique used to detect specific proteins. The Western blot process involves tissue preparation, gel electrophoresis to separate proteins, transferring proteins to a membrane, blocking the membrane, primary and secondary antibody detection to reveal targeted proteins, and analysis. Detection can be done through colorimetric, chemiluminescent, or radioactive methods. The Western blot is commonly used as a confirmatory test for HIV and hepatitis infections.

1. Presented By MADIHA AHMAD
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2. Blots are techniques for transferring DNA , RNA and
proteins onto a carrier so they can be separated, and
often follows the use of a gel electrophoresis. The
Southern blot is used for transferring DNA, the Northern
blot for RNA and the western blot for PROTEIN
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3. w
Blotting technique
Southern Blot
It is used to detect
DNA.
Northern Blot
It is used to detect
RNA.
Western blot
It is used to detect
protein
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4. A technique for detecting specific proteins separated
by electrophoresis by use of labeled antibodies.
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5. Tissue preparation
Gel electrophoresis
Transfer
Blocking
Detection
Analysis
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6. 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
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7. 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.
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8. In order to make the proteins _accessible to antibody
detection, they are moved from within
the gel onto a membrane
made of nitrocellulose or
polyvinylidene difluoride
(PVDF).
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9. The membrane is placed on top of the gel, and a stack of
filter papers placed on top of that. The entire stack is
placed in a buffer solution which moves up the paper by
capillary action, bringing the proteins with it.
Another method for transferring the proteins is called
electro blotting and uses an electric current to pull
proteins from the gel into the PVDF or nitrocellulose
membrane
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10. The membrane has the ability to bind to proteins in 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.
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11. There are two steps for the detection of the protein
Primary antibody
After blocking, a dilute solution of primary antibody
(generally between 0.5 and 5 micrograms/mL) is
incubated with the membrane under gentle agitation
The solution is composed of buffered saline
solution with a small percentage of detergent, and
sometimes with powdered milk.
The antibody solution and the membrane can be
sealed and incubated together for anywhere from 30
minutes to overnight
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12. Secondary antibody
After rinsing the membrane to remove
unbound primary antibody, the membrane is
exposed to another antibody, directed at a
species-specific portion of the primary antibody.
Several secondary antibodies will bind to
one primary antibody and enhance the signal
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14. calorimetric detection
The colorimetric detection method depends on incubation of the
western blot with a substrate that reacts with the reporter enzyme
(such as peroxidase)
Chemiluminescent detection
Chemiluminescent detection methods depend on
incubation of the western blot with a substrate that will
luminescent when exposed to the reporter on the
secondary antibody.
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15.  Radioactive detection
Radioactive labels do not require enzyme
substrates, but rather allow the placement of
medical X-ray film directly against the western
blot which develops as it is exposed to the label
and creates dark regions which correspond to
the protein bands of interest
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16. •The confirmatory HIV test employs a western blot to
detect anti-HIV antibody in a human serum sample.
Proteins from known HIV-infected cells are separated
and blotted on a membrane as above. Then, the serum
to be tested is applied in the primary antibody
incubation step; free antibody is washed away, and a
secondary anti-human antibody linked to an enzyme
signal is added. The stained bands then indicate the
proteins to which the patient's serum contains antibody.
Western blot can also be used as a confirmatory test
for Hepatitis B infection.
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17. If a protein is degraded quickly, Western blotting
won't detect it well.
This test takes longer that other existing tests
It might also be more costly
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