What is Blot?

In molecular biology blots is a technique for transferring DNA ,

RNA and proteins onto a carrier so they can be separated, and
often follows the use of gel electrophoresis.
Types OF Bolting:
Southern Blotting: Used For DNA Detection in a sample
Northern Blotting : Used For RNA Detection in a sample
Western Blotting : Used For Proteins in a sample
Northern Blotting
The northern blot is a technique used in molecular
biology to study gene expression by detection of RNA
(or isolated mRNA) in a sample. Developed by Alwnie
and his colleagues in 1979. This method was named for
its similarity to the technique known as a Southern blot.
Northern Blotting
 Applications
 Northern blots are particularly useful for determining the conditions
under which specific genes are being expressed, including which tissues in
a complex organism express which of its genes at the mRNA level.


When trying to learn about the function of a certain protein, it is
sometimes useful to purify mRNA from many different tissues or cell
types and then prepare a northern blot of those mRNAs, using a cDNA
clone of the protein of interest as the probe.

 Only mRNA from the cell types that are synthesizing the protein will
hybridize to the probe.
 Procedure
STEP 1 : ISOLATAION OF RNA :
All the target RNA’s molecules should be extracted from
the sample.
Isolate RNA from cells or tissue samples using the TRI
reagent or genelute™ kit for mammalian cells or tissues.

Step 2 : Agarose Gel Electrophoresis :

In gel electrophoresis the mixture of mRNA molecules
separated/denatured to small fragments/molecules according to their size
using an electric field.
 Agarose Gel Electrophoresis
The sample should be pushed to electrical field
through a gel that containing small pores at a
speed that are inversely proportional to the size.
As we know that DNA/RNA have negative
charge due to the sugar-phosphate backbone
will move toward positive end of the field.
Agarose Gel Electrophoresis

Agarose & Buffer : Agarose & buffer are used is a gel to conduct
electrical current.
Formaldehyde :
Formaldehyde is used to unfragment the branched RNA molecule
to simple linear one and to prevent it form coiling again.
Gel Electrophoresis
 Blotting/ Transfer
The transfer or blotting is the step in
which the mRNA from the
electrophoresis gel will be transferred
onto a nylon membrane so it may be
accessible to a probe for hybridization
and detection. The separated mRNA
bands are then blotted on chemically
reactive filter paper.
 Blotting/ Transfer
Traditionally, a nitrocellulose membrane is used, although nylon
or a positively charged nylon membrane may be used.
Nitrocellulose typically has a binding capacity of about
100µg/cm, while nylon has a binding capacity of about 500
µg/cm. Many scientists feel nylon is better since it binds more
and is less fragile.

RNA,s will be covalently link to membrane.

Blotting/ Transfer
 Hybridization/ Probe
In molecular biology hybridization means the process of
forming a double stranded nucleic acid from joining two
complementary strands of DNA (or RNA). Pre-hybridization
before hybridization blocks non-specific sites to prevent the
single-stranded probe from binding just anywhere on the
membrane.
Incubate membrane with labeled DNA or RNA probe with
target sequence.
 Hybridization/ Probe
Incubate for several hours at suitable renaturation
temperature that will permit probe to anneal to its
target sequence(s).
Probe sequence is complementary to the RNA of
interest.
Hybridization/ Probe
 Washing And Visualization
Wash Nylon from excess of probe & dry.
Place Nylon sheet over x-ray film.
X-ray film darkens where the fragments are
complementary to the radioactive probes.
 Advantages/Application
 A standard for direct study of the gene expression at the level of
mRNA.
 Detection of mRNA transcript size
DISADVANTAGE :
 Time consuming procedure .
 Use of radioactive probes.
 Detection with multiple probes is a problem.
Northern And Southern Blotting Difference
Bothe are same in protocol but some difference as below:
Character Southern Northern

Introduction Southern blotting was This method was

developed for the very first developed by the
time by the E.M. Southern and his colleagues in
in 1975. 1979.
For DNA For RNA
Function Need of Denaturation No Need
Denaturation DNA-DNA hybridization RNA-DNA hybridization
Hybridization Southern name of Northern is a misnomer

Name
Inventor