Blotting method research: · Blot analysis is a strong technique for detecting particular biomolecules in materials with a diverse ma...
Blotting method research:
·
Blot analysis is a strong technique
for detecting particular biomolecules in materials with a diverse makeup.
·
Blotting methods are used in
conjunction with gel electrophoresis, which is a resolving process for
separating DNA, RNA, and proteins.
·
It may be used on biomolecules that
can bind their corresponding ligand while adhering firmly to a support material
like nitrocellulose, nylon, or paper membrane. Before being transferred to the
membrane, the biomolecules of interest are usually sorted by size and/or
charge.
·
Edwin M. Southern invented the
technique for DNA analysis in 1975. He demonstrated that electrophoretically
fractionated DNA restriction fragments may be transferred to a solid substrate
(nitrocellulose) and identified as distinct bands after hybridization with a
complementary DNA probe.
·
Northern Blotting was coined after
the Southern blotting process was applied to RNA. Western blot analysis is the
process of transferring proteins to a membrane and detecting them using an
antibody probe.
·
All of the techniques involve a
stage in which molecules are moved from the gel to a porous membrane, which is
usually accomplished by soaking a solution through the gel and membrane with
absorbent paper.
·
Molecular hybridization with
labelled nucleic acid probes detects particular sequences in the membrane for
DNA and RNA. Labeled antibodies are used to detect the proteins.
·
The original procedure was modified
to work with radioactive probes labelled with 32P, 3H, 35S, or 125I, for
example. Other labelling methods, including as fluorescent and chemiluminescent
chemicals, have been developed since then.
Principles in general
All
three blotting procedures are quite straightforward, with four distinct steps:
1.
Separation of protein or nucleic
acid fragments in a sample by electrophoresis
2.
Immobilization of analytical probe
on paper,
3.
Binding of analytical probe to
target molecule on paper, and
4.
Visualization of bound probe
Electrophoresis
is used to separate molecules in a sample before they are transported to a
support medium or membrane that can be handled easily. This immobilises the
protein or DNA fragments, allowing for a more accurate replication of the
initial separation and easier biochemical examination.
After
being transferred to the support medium, the immobilised protein or nucleic
acid fragment is localised using probes that selectively attach to the molecule
of interest, such as antibodies or DNA. Finally, autoradiography is used to
observe the location of the probe that is attached to the immobilised target
molecule.
Procedure:
1.
Run the DNA/RNA/Protein gel to the
required length.
2.
Use a UV box to visualise the gel.
3.
Soak the gel in 0.2 N HCl until the
colour of the bromophenol blue band starts to change. (It generally takes
around 10 minutes.) Alternatively, expose for 90 seconds to a UV
transilluminator.
4.
Rinse with H2O once more.
5.
Soak for 30 minutes in 0.4N
NaOH/0.6M NaCl
6.
Create a transfer pyramid by placing
a glass plate on a support in a tray holding 1 litre of 0.5 M NaOH and covering
it with two sheets of filter paper (Whatman 3MM or other appropriate paper) so
that it contacts the solution on all sides and serves as a wick.
7.
Wet the filter paper with 0.5 M NaOH
and gently spin a glass pipette back and forth over the paper to eliminate air
bubbles.
8.
Place the filter paper on top of the
gel. Place a piece of nylon membrane on the gel that is slightly bigger than
the gel and has been prewet in 0.5 M NaOH. 4 sheets of cut-to-size prewet
filter paper Between each layer, make sure there are no air bubbles.
9.
Place a 10 cm stack of dry filter
paper or paper towels on top of the pyramid, trimmed to size. Place a glass
plate on top and a 0.5 kg weight on top.
10.
Mark the orientation of the gel and
nylon filter, for example, by cutting off a corner. Wrap strips of parafilm or
plastic wrap over the gel to prevent direct contact between the filter paper
stack and the wick, which might cause a short-circuit in the transfer
solution's flow. Allow 12 hours or more for the transfer to complete.
11.
Dismantles the pyramid with care.
12.
Remove the membrane from the
blotter. Remove the gel, whatman, and paper towels from the equation.
13.
Using 2X SSC, rinse the membrane.
Using a piece of whatman, blot dry.
14.
Bake for 1 hour in a vacuum oven
with the blot sandwiched between two pieces of dry whatman.
Blotting Techniques in Practice
Northern Blotting
Northern
blotting allows researchers to compare the expression patterns of a gene across
tissues, organs, developmental stages, stress conditions, and pathogen
infection.
When
contrasted to 'normal' tissue, the method has been used to reveal
overexpression of oncogenes and downregulation of tumor-suppressor genes in
malignant cells, as well as gene expression in the rejection of donated organs.
Southern Blotting
Southern
blotting has a variety of uses in molecular biology, including detecting RFLPs
used in the building of genomic maps and identifying one or more restriction
fragments that contain a gene or other DNA sequence of interest.
Western Blotting
The
western blot (also known as an immunoblot) is a technique for detecting
particular proteins in a tissue homogenate or extract sample. The technique was
developed in George Stark's Stanford laboratory. W. Neal Burnette coined the
term "western blot" to describe the procedure.
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