BLOTTING TECHNIQUES

Presented TO
Mam Mah Noor Khan
Presented By :
Sajida Bibi
Iqra Khalil
Sajid Ali Khan
LEARNING OUTCOME
 WHAT IS BLOTTING?
MECHANISM OF BLOTTING.
TYPES OF BLOTTING?
• APPLICATIONS OF BLOTTING.
 BLOTTING MEANS

Transfer by means of a blot.

BLOTTING is a technique for detecting DNA, RNA or proteins initially
present in a complex mixture. In this technique the molecules separated by
Gel electrophoresis

Gel electrophoresis is a method for separation and analysis of

macromolecules (DNA, RNA and proteins) and their fragments,
based on their size and charge
 GENERAL PROCEDURE

 The target molecule in the sample is separated

 Electrophoresis separated the molecules.

 Transfer to a membrane.

 Detection with a labeled probe.

Probes
A probe is a single-stranded sequence of DNA or
RNA used to search for its complementary
sequence in a sample genome.

The probe is placed into contact with the sample

under conditions that allow the probe sequence to
hybridize with its complementary sequence.
Hybridization, as related to genomics, is the process in which
two complementary single-stranded DNA and/or RNA molecules bond

together to form a double-stranded molecule. The bonding is

dependent on the appropriate base-pairing across the two single-

stranded molecules.
TYPES OF BLOTTING

There are basically 4 types of blotting:

 1) Southern blotting (DNA)
 2) Western blotting (Protein)
 3) Northern blotting (RNA)
 4) Eastern blotting (Carbohydrates and
lipid)
Southern blotting
This method is used for analysis of DNA sequences .

Southern blotting is named after Edward M. Southern.

 It involves the following steps:
Firstly, large weighted DNA is cut into small fragments by using
Restriction endonucleases
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 .
 After separating these fragments, placed a nitrocellulose sheet
over the separating gel.
Apply pressure over the membrane so that proper interaction
can occur between these two.
 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 SSC buffer and it can be
visualized on Xray film with the help of
autoradiography .
Applications:

i) It is used in the technique called RFLP

(Restriction fragment length polymorphism)

mapping.

ii) Also used in phylogenetic analysis .

iii) To identify the gene rearrangements

WESTERN BLOTTING
Western blotting is named after W. Neal Burnette.

This method is used for detection and analysis of protein in a

given sample.
A mixture of proteins is separated based on
molecular weight through gel electrophoresis.
 The gels 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 bound antibodies are then detected by
developing the film.
Applications:
Used for clinical purposes.
 used to detect low abundance specific proteins;
used for quantification of gene products
First, mRNA is extracted from cells and purified .
  Isolate this RNA on an agarose gel containing
formaldehyde as a denaturant for RNA .
Soak the gel in depurination buffer for 5–10 minutes
and wash with water .
 These RNA fragments are then transferred to
a carrier membrane. H. Aminobenzyloxymethyl  After
the filter paper
.  RNA is transcribed, it  Add is fixed to the membrane
using UV or heat.
 DNA-labeled probes for hybridization
Unbound probe is washed away and finally mRNA-
DNA hybrids are detected by X-ray film
Applications:
 i) used for screening
ii) study gene expression .
 iii) when diagnosing illness;
ESTERN BLOTTING
 Eastern blotting is given by Bogdanov.
This method is used to identify carbohydrate
epitopes including glycoconjugates and lipids
 Mostly blotted proteins after transferring onto
the membrane are analyzed for PTMs by using a
probe and hence identify carbohydrates and lipids .
. It involves the following steps:

Firstly, targeted molecules are vertically

separated by using gel electrophoresis .
 Then, these separated molecules are
transferred horizontally on the nitrocellulosic
membrane .
 After that primary antibody is added to the
solution. These antibodies are responsible for
recognizing a specific amino-acid sequence
Then wash it to remove unbound primary
antibody and add labelled secondary
antibody .
These labelled probes confirm the
molecule of interest.
i) Detection of protein modification.
ii) Used for binding studies by using
various ligands .
iii) Used to purify various phospholip.
Applications of blotting techniques
Diagnosis of infectious diseases like tuberculosis.
 Diagnosis of tumors like leukemia and lymphoma.
 Diagnosis of genetic diseases.
 In forensic medicine like paternity testing.