What is Blotting ?

 Process in detecting any macromolecule that we deal with it

 Macromolecule may be DNA, RNA or Protein

 IF we are detecting DNA we called it Southern Blotting

 IF we are detecting RNA we called it Northern Blotting

 IF we are detecting Protein we called it Western Blotting

 Northern Blotting

 Technique developed in 1977 or 1979 by J.Alwine, D. Kemp &

G. Start

 The northern blot is a technique used in molecular biology to study

gene expression by detection of RNA (or isolated mRNA) in a
sample.

 Technique based on Nucleic acid Hybridization

 This method was named for its similarity to the technique known as
a Southern blot.
 Northern Blotting (Importance)
To Study gene expression by detecting RNA
in a Sample During differentiation,
morphogenesis as well as abnormal or
diseased condition.

Detects the presence a specific mRNA in a

total RNA extract

Can determine whether a gene is

transcribed or not
 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.
 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 & Buffer : Agarose & buffer are used is a gel to conduct
electrical current.
 Formaldehyde : Formaldehyde is used to un fragment the branched
RNA molecule to
simple linear one and to prevent it form coiling again.
 Blotting/ Transfer

 IMPRINTING gel material on to the paper

 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.

 In this Blotting We use Whitman's Filter paper No. 52

 Amino Benzoxy Methyl membrane Filter Paper for Southern Blotting

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 500μg/cm. Many scientists feel
nylon is better since it binds more and is less fragile.

RNA,s will be covalently link to membrane.

Better transfer medium for RNA More binding affinity towards RNA

Transfer the content of gel onto the paper is takes place by through
capillary action
 After transferring the content of gel (RNA) from gel to the paper
 We Exclude everything out and take membrane out. Put the
membrane into a solution containing
 Probes.
 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.
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.

 Western blot

 Western blot is the analytical technique used in molecular biology, immunogenetics and other
molecular biology to detect specific proteins in a sample of tissue homogenate or extract.

 Western blotting is called so as the procedure is similar to Southern blotting. While Southern blotting
is done to detect DNA, Western blotting is done for the detection of proteins.

 Western blotting is also called protein immunoblotting because an antibody is used to specifically
detect its antigen.

Principle

 Western blotting technique is used for identification of particular protein from the mixture of protein.

 In this method labelled antibody against particular protein is used identify the desired protein, so it is
a specific test.

 Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
 The technique consists of three major processes

1.Separation of proteins by size (Electrophoresis).

2.Transfer to a solid support (Blotting)
3.Marking target protein using a proper primary and secondary antibody to visualize (Detection).
 Procedure/Steps

1.Extraction of protein

2.Gel electrophoresis: SDS PAGE

3.Blotting: electrical or capillary blotting

4.Blocking: BSA

5.Treatment with primary antibody

6.Treatment with secondary antibody( enzyme labelled anti Ab)

7.Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-

nitro phenyl phosphate which give color.
  Extraction of Protein

 Cell lysate is most common sample for western blotting.

 Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as
tissue preparation.
 To prevent denaturing of protein protease inhibitor is used.
 The concentration of protein is determined by spectroscopy.
 When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing
glycerol which helps to sink the sample in well.
 Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.

  Samples may be taken from whole

tissue or from cell culture. In most cases,
solid tissues are first broken down
mechanically using a blender.

 It should be noted that bacteria, virus or

environmental samples can be the
source of protein and thus Western
blotting is not restricted to cellular
studies only.
  Gel electrophoresis

 The proteins of the sample are separated using gel electrophoresis. Separation of proteins may
be by isoelectric point, molecular weight, electric charge, or a combination of these factors

 The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel
electrophoresis.

 The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or
combination of these all.

 The small size protein moves faster than large size protein.

 Protein are negatively charged, so they move toward positive (anode) pole as electric current is
applied.
 Blotting/ Transfering

 The nitrocellulose membrane is placed on the gel.

The separated protein from gel get transferred to
nitrocellulose paper by capillary action. This type of
blotting is time consuming and may take 1-2 days

 For fast and more efficient transfer of desired protein

from the gel to nitrocellulose paper electro-blotting
can be used.

 In electro-blotting nitrocellulose membrane is

sandwich between gel and cassette of filter paper
and then electric current is passed through the gel
causing transfer of protein to the membrane.
 Blocking

 Blocking is very important step in western

blotting.

 Antibodies are also protein so they are likely to

bind the nitrocellulose paper. So before adding
the primary antibody the membrane is non-
specifically saturated or masked by using casein
or Bovine serum albumin (BSA).
 Treatment with Primary
Antibody

•The primary antibody (1° Ab) is

specific to desired protein so it
form Ag-Ab complex

Treatment with secondary

antibody

•The secondary antibody is

enzyme labelled. For eg.
alkaline phosphatase or
Horseradish peroxidase (HRP)
is labelled with secondary
antibody.

•Secondary antibody (2° Ab) is

antibody against primary
antibody (anti-antibody) so it
can bind with Ag-Ab complex.