Blotting

BLOTTING
• Blotting is a technique of transferring proteins, DNA
and RNA onto a solid support generally nylon or
nitrocellulose membrane, so they can be separated
and often follows the use of gel electrophoresis.
• Identifying and measuring specific proteins in
complex biological mixtures, such as blood used in
diagnostic practice. Identification of abnormal genes
in DNA used in clinical research.
• These are used to identify protein and nucleic acid
sequences used in molecular biology.
Types of blotting
 Southern Blotting
• Professor Sir Edwin Southern developed this
technique in 1975.
• He won Lasker Award of clinical medical research
prize for this method of finding DNA sequence.
• This method is routinely used in molecular biology
for detection of a specific DNA sequence in DNA
samples. The DNA detected can be a single stretch
of gene or it can be a larger piece of DNA.
• It combines agarose gel electrophoresis for size
separation of DNA method to transfer the size
separated DNA to a filter membrane for probe
hybridization.
 Principle of Southern blotting

Southern blotting is based on the principle of

separation of DNA fragments by gel electrophoresis
followed by the identification by labeled probe
hybridization. The DNA fragments are separated
based on their size and charge during
electrophoresis.
 Steps in Southern blotting
• The DNA to be analyzed, such as the total DNA of an
organism , is digested with a restriction enzyme.
• The complex mixture of fragments is subjected to gel
electrophoresis to separate the fragments according to size.
• The restriction fragment present in the gel are denatured with
alkali and transferred onto nitrocellulose or nylon membrane
by blotting.
• The filter is incubated under hybridization conditions with a
specific radio labelled DNA probe. The probe hybridizes to
the complementary DNA restriction fragment.
• Excess probe is washed away and the probe bound to filter is
detected by autoradiography.
 Applications
• It is used in gene discovery, mapping, evolution
and development studies.
• It is used to ensure that a particular section of
DNA is successfully incorporated into genome of
host organism.
• Analyze the genetic pattern present in person’s
DNA.
• Analyze restriction digestion fragment of DNA.
 Northern blotting
• It is used to study gene expression by detection of specific RNA sequence in a
sample.
• It was developed by James Alwine and George Stark in 1979 at Stanford
university.
Steps
• RNA is isolated from several biological samples.
• Samples are loaded on gel and the RNA samples are separated according to
their size on an agarose gel.
• The gel is then blotted on Diazobenzyloxymethyl membrane.
• The membrane is placed in a dish containing hybridization buffer with a
labeled probe that will hybridize to RNA.
• The membrane is washed to remove unbound probe.
• The labeled probe is detected via autoradiography.
 Applications
• Study m-RNA transcript size.
• Study RNA degradation.
• Study RNA half life.
• Study RNA splicing.
• Study gene expression.
 Western blotting
• It is immunoblotting technique which rely on the
specificity of binding between a molecule of interest
(protein) and probe to allow detection of the protein in
a mixture of many other similar molecules.
• Probe used here is antibody
Steps:
• A protein sample is subjected to electrophoresis on an
SDS Polyacrylamide gel.
• Electroblotting transfers the separated proteins from
the gel to the surface of nitrocellulose membrane.
 Steps
• Any secondary antibody specific for the protein of
interest is added to the nitrocellulose membrane.
• Removed the unbound antibodies and then added
radiolabelled or enzyme linked secondary antibody
to visualise the protein-Ab1-Ab2 complex
 Applications
• It is used in HIV confirmatory test.
• It is used for testing of mad cow disease.
 South western blot

• South western blot was discovered by B. Bowen and J.

Steinberg, is used to identify and characterize DNA binding
proteins.
• It includes two techniques DNA detection by Southern
blotting and protein detection by Western blotting.
• In this proteins are separated on a polyacrylamide gel and
sodium dodecyl sulphate, renatured by removing SDS in the
presence of urea and blotted onto nitrocellulose membrane by
diffusion.
• The genomic DNA region of interest is digested by restriction
endonucleases to produce appropriate fragment size which are
labelled at ends and allowed to bind to proteins.
• The bound DNA is eluted from protein DNA complex and
analyzed on polyacrylamide gel electrophoresis.
• This allows the purification of bound DNA fragments and
improve protein mediated cloning of DNA.
 Eastern blot
• It is a biochemical technique used to analyze
protein post translational modifications
including the addition of lipids, phosphates, and
glycoconjugates. It is often used to detect
carbohydrate epitopes.
• Proteins blotted from SDS PAGE gel on to a
nitrocellulose membrane. Transferred proteins
are analyzed for PTM .
 Far eastern blot
• It is a technique for the analysis of lipids secreted by
high performance thin layer chromatography. In this
lipids are transferred from HPTLC to a PVDF
membrane for further analysis.
• It was developed in 1994 by Taki at the Tokyo
medical and dental university, Japan.
• It allows the purification of glycosphingolipids,
phospholipids and structural analysis of lipids in
conjugation with direct mass spectrometry.
 Dot Blot
• A dot blot technique is used to detect proteins.
• In this sample is applied directly to membrane as a
single spot.
• It involves spotting 1 to 2 µl sample on nitrocellulose
membrane.
• Samples can be in the form of tissue culture, blood
serum.
• It is incubated with primary antibody followed by
secondary labelled antibody with alkaline phosphate.
• After binding incubated with chemiluminescent
substrate.
 Northwestern blot
• The northwestern blot, also known as
the northwestern assay, is a hybrid analytical
technique of the western blot and the
northern blot, and is used in molecular biology
to detect interactions
between RNA and proteins.
• The northwestern blot combines the two
techniques, and specifically involves the
identification of labeled RNA that interact with
proteins that are immobilized on a similar
nitrocellulose membrane.
DNA Sequencing
 NGS
• Next-generation sequencing (NGS) is a
modern method of analyzing genetic material
that allows for the rapid sequencing of large
amounts of DNA or RNA. Unlike traditional
sequencing techniques, NGS can
simultaneously sequence millions of small
fragments of DNA, enabling researchers to
expand the scale and discovery power of their
genomic studies.
 APPLICATIONS
• Resequencing of the human genome is being performed to identify
genes and regulatory elements involved in pathological processes.
• NGS has also provided a wealth of knowledge for comparative
biology studies through whole-genome sequencing of a wide
variety of organisms.
• NGS is applied in the fields of public health and epidemiology
through the sequencing of bacterial and viral species to facilitate
the identification of novel virulence factors.
• Additionally, gene expression studies using RNA-Seq (NGS of
RNA) have begun to replace the use of microarray analysis,
providing researchers and clinicians with the ability to
visualize RNA expression in sequence form.