The transfer of nucleic acid fragments or proteins
from gel to a suitable solid support is known as “Blotting”
Or Biochemical technique in which macromolecules
separated on a gel are transferred to a nylon membrane or sheet of paper,
thereby immobilizing them for furthur analysis.
Electro-Blotting:
an important method to transfer mainly proteins from polyacrylamide gel on to
nitrocellulose or other carier membrane. A technique for the electrophoretic transfer of DNA, RNA or
protein to a suitable membrane. The method most commonly used for the
electrotransfer of proteins to nitrocellulose is that reported by Towbin et al. (1979). This technique
was patented in 1989 by William J.
Littlehales under the title "Electroblotting
technique for transferring specimens from a polyacrylamide electrophoresis or
like gel onto a membrane." Transfer of the proteins can be carried out
using several methods such as vacuum, capillary or electric field.
Electroblotting is by far the most wide-spread technique which utilizes either
vertical buffer tanks or semi-dry blotting
Tank-Blotting:
The gel & blotting membrane are clamped fram between filter paper &
fiber pads. Typically transfer time is overnight, only little transfer buffer
is required & transfer time is very short.
Capillary-Blotting:
Traditional method for transfer of nucleic acids
- Membrane size up
to 20 x 25 cm
- Overnight
transfer
- Easy to use
Method for transfer of nucleic acids (especially RNA) from
agarose gels onto a membrane. This procedure usually takes up to 12 hours (over
night) to complete.
Easy to set up assembly that works without power or vacuum source.
This method is based on the movement of buffer from a reservoir
through the gel and the blotting membrane to a stack of dry blotting paper by
capillary force. The molecules are carried to the blotting membrane on which
they are adsorbed.
Vaccum-Blotting:
Dot & Slot Blotting:
Some of the commercially available blotting products
are:
Fastblot B33/34;
B43/44; B64
Tankblot Tankbot
Ecomini
Vacuum-Blot
Cappilary Blotting
Dot Blot 96/ System
Hybrit Slot
The convertible.
Blot is made in spot/
stain on membrane. Blotting technique
are of various types depending on the targets in the hybridization experiments
for their specific detection. Commonly are:
Dot Blotting [For
DNA/RNA]
Northern Blotting [For
RNA]
Southern Blotting [For
DNA]
Western Blotting [For
Protein]
Dot
blotting
A modification of Southern & Northern blotting
techniques where nucleic acids are directly spotted on to the filters and not
subjected to electrophoresis. The hybridization procedure is the same as in
original blotting techniques.
Sample DNA’s from several tissue/ individual can be
tested in a single test run. Dot blot are useful in detecting the presence of
the sequence being transferred in a number of suspected transgenic individuals
or in different tissues of a single individuals
Steps
involve:
Sample DNA/RNA from
different individual/tissues are transferred on to a nitrocellulose filter in
form of dots;
Denature the DNA &
backed filter at 80Âșc to fix the DNA firmly on to the filter, that prevent
non-specific binding of the probe to the filter;
The filter is then
treated with the appropriate radioactive ssDNA probe under condition favoring
hybridization;
Wash repeatedly filter
to remove the free probe;
Dots having appropriate
DNA/RNA sequence will hybridize with the radioactive probes;
The hybridization
probes are detected by autoradiography; this denotes the individual/ tissue in
which the DNA/RNA sequence corresponding to the probe is represented.
Northern
Blotting
The technique for the specific identification of RNA
molecules is known as Northern Blotting. This was developed in 1977 by “Alwine
et al” at Stanford University.
RNA is separated by RNA gel electrophoresis
subsequent transfer to membrane, hybridization with probe & finally
detection through autoradiography. RNA molecules don’t easily bind to
Nitrocellulose paper/ Nylon membrane. Blot transfer of RNA molecule is carried
out by using a chemically reactive paper prepared by diazotization of
aminobezyloxymethyl to create diazotibenzyloxymethyl (DBM) paper. The RNA can
be conventionally bound to DBM paper.
Steps
involve:
Sample target ;
RNA isolation;
Gel electrophoresis of
RNA;
Transfer to membrane;
Probe preparation;
Cross-linking of RNA to
membrane;
Pre-hybridization;
Hybridization;
Post-hybridization;
Signal detection.
Advantages:
Widely accepted &
well regarded method;
A straight forward
method;
Often used as a
confirmation or check;
Versatile protocol as
it can allow the usage of many types of probes, including radiolabeled,
non-radiolabeled; invitro-transcribed RNA & even oligo-nucleotides such as
Primers.
Disadvantages:
Often radioactivity is
used, which prevents ease of performing its use and disposal;
The whole process takes
long time;
If RNA sample are even
slightly degraded by RNases, the quality of the data and quantization of
expression is quite negatively affected.
Application:
Detection of mRNA
transcripts size.
Study RNA degradation
Study RNA splicing, can
detect alternatively spliced transcripts;
Study internal
ribosomal entry site;
Often used to confirm
& check transgenic.
Southern
Blotting
The technique for DNA separation according size by
gel electrophoresis is known as “Southern Blotting” where gel denatures in to
single stranded molecules by treatment with alkali, neutralization & transferred
to a hybridization method by using a high salt concentration buffer. DNA is
then irreversibly bound to the membrane either by heat treatment/ UV cross
linking. Thus ssDNA targeted molecules are available on the filter for
hybridization with a labeled ssDNA probe.
This technique, devised
by Ed Southern in 1975, is a
commonly used method for the identification of DNA fragments that are
complementary to a know DNA sequence. Southern hybridisation, also called Southern blotting, allows a comparison between the genome of a particular organism
and that of an available gene or gene fragment (the probe).
It can tell us whether an organism contains a particular gene, and provide
information about the organisation and restriction map of that gene.
In Southern blotting, chromosomal DNA is isolated from the organism of interest, and
digested to completion with a restriction endonuclease enzyme. The restriction
fragments are then subjected to electrophoresis on an agarose gel, which
separates the fragments on the basis of size.
DNA fragments in the gel
are denatured (i.e. separated into single strands) using an alkaline solution.
The next step is to transfer fragments from the gel onto nitrocellulose filter
or nylon membrane. This can be performed by electrotransfer (electrophoresing
the DNA out of the gel and onto a nitrocellulose filter), but is more typically
performed by simple capillary action.
In this system, the
denatured gel is placed onto sheet(s) of moist filter paper and immersed in a
buffer reservoir. A nitrocellulose membrane is laid over the gel, and a number
of dry filter papers are placed on top of the membrane. Bycapillary action,
buffer moves up through the gel, drawn by the dry filter paper. It carries the
single-stranded DNA with it, and when the DNA reaches the nitrocellulose it
binds to it and is immobilised in the same position relative to where it had
migrated in the gel.
The DNA is bound
irreversibly to the filter/membrane by baking at high temperature
(nitrocellulose) or cross-linking through exposure to UV light (nylon).
Southern blotting has became a routine technique in
the analysis of gene organization, the identification & cloning of specific
sequences.
Steps
involve:
DNA digestion & electrophoresis;
DNA blotting
Hybridization
Post-hybridization
Autoradiography
Advantages
An invaluable method in gene analysis
For confirmation of DNA cloning results;
Forensic applied to detect minute quantification for
DNA.
Western
Blotting
The technique identifies proteins involving transfer
of electrophorosed protein bands from polyacrylamide gel to nylon or
nitrocellulose membrane, detected by specific protein-ligand interaction [i.e.
Antibody/ Lectin]
Western blot identify the location of a specific
protein after it has been separated by SDS-PAGE. First a primary antibody that
recognizes one epitop binds to the protein of interest. The location of the
primary antibody is visualized by adding a secondary antibody conjugated to a
detection system.
Steps involve:
Ă Separation
of protein by Gel-electrophoresis
Ă Transfer
protein to Nitrocellulase membrane so, the protein retain the same pattern of
separation they had on the get
Ă Incubate
the blot with a generic protein to binds to any remaining sticky place on the
nitrocellulose;
Ă Photograph,
the location of the antibody, colorless substrate attached enzyme converted to colorless
product.
In this technique it doesn’t matter whether the
protein has been synthesized invivo or invitro, This technique explains the
content of protein accumulated in cells and rate of synthesis explained by
Radioimmune precipitation assay there is establishment of negative charge on
side gel while positive on nitrocellulose membrane. The protein binds better to
nitrocellulose membrane at low pH and no air bubbles between nitrocellulose
membrane and gel. The primary antibody specific to protein form
antibody-protein complex with protein of interest. The protein gel stained with
KCl so, form precipitation with an SDS-PAGE gel.
The primary antibody obtained from rabbit antisera
dilution with non-fat dry instant milk. The secondary antibody is goat
anti-rabbit, antibody against 1st antibody. The reaction usually run
out in about 1 hours.
