BLOTTING IS A METHOD TRANSFER DNA FRAGMENT FROM FEL TO MEMBRANE

1. Blotting: Southern, Northern and
Western Blotting
Mrs. Praveen Garg
VITS College, Satna

2. INTRODUCTION
• Blotting techniques are used to identify proteins and
nucleic acid sequences.
• They have been developed to be highly specific and
sensitive technique.
• It become important tools in both molecular biology and
clinical research.
• This process can be done after the gel electrophoresis, by
transferring the molecules from the gel onto the surface of
blotting membrane or solid support.
• These transferring molecules visualized by using different
stain Ethidium bromide, Crystal violet, Safranine and
Ossmium tetroxide etc

3. TYPES OF BLOTTING
There are basically 4 types of blotting:
• Southern blotting
• Western blotting
• Northern blotting
• Eastern blotting

5. SOUTHERN BLOTTING
• Southern blotting is named after Edward M. Southern
in 1975.
• This method is used for analysis of DNA fragments in a
complex mixture.
• Nitrocellulose membrane used in this method for
transferring the band.
• In this method, DNA-DNA hybridization take place.
• It is also called as southern hybridization

6. METHODOLOGY
• Firstly, large weighted DNA is cut into small fragments by
using Restriction endonucleases enzyme.
• Then, these fragments are electrophoresed on separating
gel so that they can separate according to their size.
• If DNA fragments are much larger in size so firstly the gel
should be treated with HCl, causes depurination of DNA
fragments.
• The separated DNA molecule are denatured by alkali
treatment and placed a nitrocellulose membrane over the
separating gel.
• All DNA fragments present on gel is transferred to the
membrane.

7. • After that the membrane is exposed to ultraviolet
radiation so that the fragments are permanently
attached to the membrane.
• Then the membrane is exposed to hybridization probe.
But the DNA probe is labeled so that it can easily
detect, when the molecule is tagged with a chromogenic
dye.
• After hybridization process, excess probe is washed
away by using buffer and it can be visualized on X-ray
film with the help of autoradiography.

9. APPLICATIONS
• It is used in the technique called RFLP (Restriction fragment
length polymorphism) mapping.
• Also used in phylogenetic analysis.
• To identify the gene rearrangements .
• It is important for gene cloning.

10. • The northern blot technique was developed in 1977 by
James Alwine.
• The northern blot or RNA blot, is a technique used
in molecular biology to study gene expression by detection
of RNA (or isolated mRNA) in a sample.
• Northern blotting involves the use of electrophoresis to
separate RNA samples by size.
• Its detection can be done with a hybridization
probe complementary to part of or the entire target
sequence.
• The term 'northern blot' actually refers to the capillary
transfer of RNA from the electrophoresis gel to the blotting
membrane.
NORTHERN BLOTTING

12. • Blotting procedure starts with extraction of total RNA
from a homogenized tissue sample or from cells.
• Eukaryotic mRNA can then be isolated through the use
of oligo (dT) cellulose chromatography to isolate only
those RNAs with a poly(A) tail.
• RNA samples are then separated by gel electrophoresis.
• Since the gels are fragile and the probes are unable to
enter the matrix, the RNA samples, now separated by
size, are transferred to a nylon membrane through a
capillary or vacuum blotting system.
METHODOLOGY

13. • A nylon membrane with a positive charge is the most
effective for use in northern blotting since the negatively
charged nucleic acids have a high affinity for them.
• The transfer buffer used for the blotting usually
contains formamide.
• Once the RNA has been transferred to the membrane, it is
fixed to the membrane by UV light or heat.
• After a labeled probe has been hybridized to the RNA on
the membrane.
• The membrane is washed to ensure that the probe has
bound specifically.
• The hybrids are then detected by X-ray film and can be
quantified by densitometry.

14. • Northern blotting allows to observe a particular gene's
expression pattern between tissues, organs,
developmental stages, pathogen infection.
• The technique has been used to show over expression of
oncogenes and downregulation of tumor-suppressor
genes in cancerous cells when compared to 'normal'
tissue.
• The variance in size of a gene product can also indicate
deletions or errors in transcript processing.
• By altering the probe target used along the known
sequence it is possible to determine which region of the
RNA is missing.
APPLICATIONS

15. • The western blot or western blotting, is a widely used
analytical technique in molecular biology and
immunogenetics.
• It detect specific proteins in a sample of tissue homogenate or
extract.
• The sample undergoes protein denaturation, followed by gel
electrophoresis.
• Western blotting is also called protein immunoblotting
because an antibody is used to specifically detect its antigen.
WESTERN BLOTTING

16. The technique consists of three major processes:
• Separation of proteins by size (Electrophoresis).
• Transfer to a solid support (Blotting)
• Marking target protein using a proper primary and secondary
antibody to visualize (Detection).

18. • Electrophoresis used to separate proteins according to their
electrophoretic mobility which depends on charge, size of
protein molecule and structure of the proteins.
• Proteins are moved from within the gel onto a membrane made
of Nitrocellulose (NC) or Polyvinylidene difluoride (PVDF).
• Proteins combine with nitrocellulose membrane based on
hydrophobic interaction (Blotting).
• For detection of the proteins primary antibody and enzyme
conjugated secondary antibody are used.
• On addition of substrate, a substrate reacts with the enzyme that
is bound to the secondary antibody to generate colored
substance, namely, visible protein bands.

19. • In this technique a mixture of proteins is separated based on
molecular weight, through gel electrophoresis.
• These gel are then transferred to a membrane producing a
band for each protein.
• The membrane is then incubated with labels antibodies
specific to the protein of interest.
• The unbound antibody is washed off.
• The bound antibodies are then detected by developing the
film.
• As the antibodies only bind to the protein of interest, only one
band should be visible.
• The thickness of the band corresponds to the amount of
protein present.

20. • It utilizes SDS-PAGE (Sodium dodecyl sulphate
polyacrylamide gel electrophoresis) gel electrophoresis.
• It can use nitrocellulose membrane and nylon membrane.
• After hybridization, antigen-antibody complex is formed.
• If the protein is bound by a radioactive antibody, its position on
the blot can be determined by exposing the membrane to a
sheet of X-ray film, a procedure called autoradiography.

21. • Identification of a specific protein in a complex mixture of
proteins.
• In this method, known antigens of well-defined molecular weight
are separated by SDS-PAGE and blotted onto nitrocellulose.
• The separated bands of known antigens are then probed with the
sample suspected of containing antibody specific for one or more
of these antigens.
• Reaction of an antibody with a band is detected by using either
radiolabeled or enzyme-linked secondary antibody that is specific
for the species of the antibodies in the test sample.
• Estimation of the size of the protein as well as the amount of
protein present in the mixture.
APPLICATION

22. THANK YOU