Essentials of Anatomy & Physiology, 4th Edition

Martini / Bartholomew

BLOTTING TECHNIQUES

7 The Muscular
System
By
Dr. R. A. Siddique
®
PowerPoint Lecture Outlines
prepared by Alan Magid, Duke University
 Preparation of probes

• Synthesis of uniformly labeled double-

stranded DNA probes
• Preparation of single-stranded probes
• Labeling the 5 and 3 termini of DNA
Synthesis of double-stranded DNA probes

- Nick translation of DNA

- Labeled DNA probes using random
oligonucleotide primers
Nick translation
 Preparation of single-stranded probes
• Synthesis of single-stranded DNA probes
using bacteriophage M13 vectors.

• Synthesis of RNA probes by in vitro

transcription by bacteriophage DNA-
dependent RNA polymerase.
 In vitro
transcription
Labeling the 5 and 3 termini of DNA
• Labeling the 3 termini of double-stranded
DNA using the Klenow fragment of E. coli
DNA polymerase I. (lack of 5’  3’
exonuclease activity)
• Labeling the 3 termini of double-stranded
DNA using bacteriophage T4 DNA polymerase.
• Labeling the 5 termini of DNA with
bacteriophage T4 polynucleotide kinase.
T4 polynucleotide kinase activity
 Non-isotope labeling
• Digoxigenin-11-dUTP (DIG-dUTP) labeling
- DNA labeling
- Oligonucleotide labeling
- RNA labeling
PCR Labeling, Random Primed Labeling,
and RNA Labeling
 What is blotting?
 Blots are techniques for transferring DNA , RNA
and proteins onto a carrier so they can be
separated, and often follows the use of a gel
electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA and
the western blot for PROTEIN.
 TYPES OF BLOTTING TECHNIQUES

Blotting technique

Southern Blot Northern Blot Western blot

It is used to detect DNA. It is used to detect RNA. It is used to detect protein.
Comparison of Southern, Northern, and
Western analyses of Gene X
 SOUTHERN BLOTTING
• Professor Sir Edwin Southern,
Professor of Biochemistry and
Fellow of Trinity developed this
method in 1975.
• Southern won the Lasker Award
for Clinical Medical Research
prize for the method of finding
specific DNA sequences he
developed this procedure at
Edinburgh University more than 30
years ago. The technique is
known as DNA transfer or
'Southern blotting'

Professor Sir Edwin Southern

Cont….
• This method Involves separation, transfer and
hybridization.

• It is a method routinely used in molecular biology for

detection of a specific DNA sequence in DNA samples. The
DNA detected can be a single gene, or it can be part of a
larger piece of DNA such as a viral genome.
Cont….
• Southern blotting combines agarose gel electrophoresis for
size separation of DNA with methods to transfer the size
separated DNA to a filter membrane for probe
hybridization.

• The key to this method is Hybridization.

• Hybridization - Process of forming a double-stranded DNA

molecule between a single-stranded DNA probe and a
single-stranded target patient DNA.
 PRINCIPLE
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.

3. The probe is added to the matrix to bind to the

molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected corresponds to

the location of the immobilized target molecule.
Flow chart of Southern hybridization
Preparing the samples and running the gel

Southern transfer

Probe preparation Isotope

Non-isotope
Prehybridization

Hybridization

Post-hybridization washing

Signal detection
APPARATUS
Whatman 3MM paper nitrocellulosemembrane
Capillary blotting apparatus
 Example of Transfer
Upward Capillary Transfer
Weight
Glass Plate
Paper towels
Membrane (nylon or Whatman 3MM paper
)
nitrocellulose

Whatman
3MM paper Gel

Transfer buffer
 weight  tight connection

DNA eluted
Buffer drawn from the gel
from a by the
reservoir moving
passes stream of
through the buffer is
gel into a deposited
stack of paper onto a
towels membrane
 Steps in southern blotting
1. Digest the DNA with an
appropriate restriction
enzyme.

2.The complex mixture of

fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.
 Cont….
3.The restriction fragments
present in the gel are
denatured with alkali and
transferred onto
4. a nitrocellulose filter or nylon
membrane by blotting.

• This procedure preserves the

distribution of the fragments in
the gel, creating a replica of the
gel on the filter.
Cont….

5.The filter is incubated under

hybridization conditions
with a specific radiolabeled
DNA probe.

• The probe hybridizes to the

complementary DNA
restriction fragment.
Cont….

6.Excess probe is washed away and the

probe bound to the filter is detected by
autoradiography, which reveals the
DNA fragment to which the probe
hybridized.
 APPLICATIONS
• Southern blots are used in gene discovery , mapping,
evolution and development studies, diagnostics and
forensics (It is used for DNA fingerprinting, preparation of RFLP maps)

• identification of the transferred genes in transgenic individuals, etc.

 APPLICATIONS
• Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to measure
relative amounts in different samples.

• Southern blot is used to detect the presence of a particular

bit of DNA in a sample

• analyze the genetic patterns which appear in a person's

DNA.
Detection of an RFLP by
Southern blotting
Detection of the sickle-cell globin gene by
Southern blotting
Checking of the gene knockout mice
 Northern Blotting
Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark at
Stanford University (1979) and was named such by
analogy to Southern blotting

James Alwine George Stark

The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel

Northern transfer

Probe preparation Isotope

Non-isotope
Prehybridization

Hybridization

Post-hybridization washing

Signal detection
 Steps involved in Northern
blotting

1. RNA is isolated from several

biological samples (e.g.
various tissues, various
developmental stages of same
tissue etc.)
* RNA is more susceptible to
degradation than DNA.
Cont……

2. Sample’s are loaded on

gel and the rna samples
are separated
according to their size
on an agarose gel .

• The resulting gel

following after the
electrophoresis run.
Cont……

3. The gel is then

blotted on a nylon
membrane or a
nitrocellulose
filter paper by
creating the
sandwich
arrangement.
Cont……

4. The membrane is placed in a

dish containing
hybridization buffer with a
labeled probe.
• Thus, it will hybridize to the
RNA on the blot that
corresponds to the sequence
of interest.

5. The membrane is washed to

remove unbound probe.
Cont……
6. The labeled probe is detected
via autoradiography or via
a chemiluminescence
reaction (if a chemically
labeled probe is used). In
both cases this results in the
formation of a dark band
on an X-ray film.
• Now the expression patterns
of the sequence of interest
in the different samples can
be compared.
 Northern Steps
1. Isolate RNA & treat with formaldehyde.

2. Electrophorese RNA in denaturing agarose gel (has

formaldehyde). Visualize RNA in gel using Ethidium bromide
stain and photograph.

3. Transfer single-stranded RNA to nitrocellulose or nylon

membrane. Covalently link RNA to membrane.

4. Incubate membrane (RNA immobilized on membrane) with

labeled DNA or RNA probe with target sequence.

5. Development.

42
 Step 1
Isolate RNA:
-To detect rare mRNA, isolate the poly A+ mRNA.
-RNA is both biologically and chemically more labile than DNA.
Thus eliminate RNases.

Step 2
Electrophoresis:
-Performed in formaldehyde agarose gel to prevent RNA
from folding on itself.
-Stain with EtBr to visualize the RNA bands.

43
 Step 3
-Transfer single-stranded RNA to nitrocellulose or nylon
membrane:

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.

-Covalently link RNA to membrane:

UV cross linking is more effective in binding RNA to the

membrane than baking at 80C.
44
 Step 4 & 5
-Prehybridize 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:
Probe could be 32P, biotin/streptavidin or a bioluminescent
probe.

-Autoradiography:
Place membrane over X-ray film.
X-ray film darkens where the fragments are complementary to
the radioactive probes.

45
Agarose gel & Membrane & Autoradiography

Sobel, R., Dubitsky, A.,

Brickman, Y. (2002) Genetic
Engineering News 23, 2.
46
 Northern Application

• 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.

• For instance:

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.
47
 APPLICATIONS
• A standard for the study of gene expression
at the level of mRNA (messenger RNA
transcripts)

• Detection of mRNA transcript size

• Study RNA degradation

• Study RNA splicing

• Study RNA half-life

• Often used to confirm and check transgenic /

knockout mice (animals)
 Summary
Southern Northern
• DNA on membrane. • RNA on membrane.
• Digest DNA. • No need to digest DNA.
• Convert dsDNA to • Denature “folded” RNA
ssDNA. with formaldehyde.
• Probe with DNA or RNA. • Probe with DNA or RNA.

49
 Disadvantage of Nourthern
plotting
1.The standard northern blot method is
relatively less sensitive than nuclease
protection assays and RT-PCR

2. Detection with multiple probes is a problem

3. If RNA samples are even slightly degraded by

RNases, the quality of the data and
quantitation of expression is quite
negatively affected.
 Western Blotting
“Immunoblotting”
= electrophoretic transfer of proteins from
gels to membranes

Towbin H W. Neal Burnette

 WB: Definition
 Blotting is the transfer of separated proteins
from the gel matrix into a membrane, e.g.,
nitrocellulose membrane, using electro- or
vacuum-based transfer techniques.
Towbin H, et al (1979). "Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications.". Proc Natl
Acad Sci U S A. 76 (9): 4350–4354

The name western blot was given to the technique by W. Neal

Burnette
 Applications & Advantages
Applications:
To determine the molecular weight of a protein
(identification)
To measure relative amounts (quantitation) of
the protein present in complex mixtures of proteins
that are not radiolabeled (unlike immunoprecipitation)
Advantages:
WB is highly sensitive technique
As little as 1-5 ng of an average-sized protein can be
detected by WB
 Flow chart of Western blotting
Electrophoresing the protein sample

Assembling the Western blot sandwich

Transferring proteins from gel to nitrocellulose paper

Staining of transferred proteins

Blocking nonspecific antibody sites on the nitrocellulose paper

Probing electroblotted proteins with primary antibody

Washing away nonspecifically bound primary antibody

Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and

formation of a diaminobenzidine (DAB) precipitate

Photographing the immunoblot

 Western blotting

The main steps of blotting technique in a

chronological order will be as follows:
• Blocking
• Probing with the specific antibody(ies)
• Wash
• Detection
• Washing
• X-ray (Gel Documentation System)
Electrophoretic Transfer: An Overview

Important Issue:
Where to put the gel and the membrane relative to the
electroblotting transfer electrodes?
 Direction of Transfer

 Perpendicularly from the direction of travel of proteins

through the separating gel

Gel Probe with specific Ab

Membrane

Dr. Azhar Chishti

Factors Affecting Transfer Efficiency

1. The Composition of the gel

2. Whether there is complete contact of
the gel with the membrane
3. The position of the electrodes
4. The transfer time
5. The size & composition of proteins
6. The field strength
7. The presence of detergents
 WB Procedure; Briefly…
2 1

3 4
www.bio.davidson.edu/.../method/West
Direct Detection Method
Indirect Detection Method
WB: examples of used
substrates
Chemiluminescent substrates
Enhanced ChemiFluoresenct (ECF) WB
Detection
Essentials of Anatomy & Physiology, 4th Edition
Martini / Bartholomew

7 The Muscular
Western Blotting Procedure; Illustrated
System

PowerPoint® Lecture Outlines

prepared by Alan Magid, Duke University
Steps of WB
Steps of WB
 Why to block?
Steps of WB
To increase sensitivity
To prevent nonspecific signal
 Blocking of Blot

Several measures should be followed to

decrease the nonspecific reactions to a
minimum, i.e., increasing the signal to noise
ratio.

Blocking step is the incubation of the

membrane with solution containing BSA
or fat-free milk or casein for a sufficient time
with shaking.
 Steps of WB

For Direct Transfer, choices are:

Dr. Azhar Chishti

 Primary Antibody labeling

• The immobilized proteins on the surface

of the membrane can be detected using
a specific, labeled antibody.

• Labeling of the antibody can be

performed using a radioactive or non-
radioactive method.
 Primary Antibody probing

• The blot is first incubated with a primary

antibody followed by the addition of a labeled
secondary antibody that has species
specificity for the primary one.
• For example, probing of the membrane
using mouse primary antibody and anti-
mouse secondary antibody.
Steps of WB
Steps of WB
Analysis of protein samples by SDS polyacrylamide-gel
electrophoresis and Western blotting

Protein bands
detected by
specific antibody

SDS-PAGE Western blot

 Detection and interpretation

• A prestained MW standard is included in a

separate lane during electrophoresis to
allow the identification of the MW of the
target protein.

• Similar to the analysis of electrophoresis

results on a gel, the data on the membrane
can be quantitatively analyzed using gel
documentation system.
 Detection and interpretation (continue)

• Quantification of a specific protein band

can be achieved by densitometry and
integrating the areas under the peaks.

• Several gel documentation systems are

commercially available that can be useful
for analysis of results from the gel or
membranes.
 Comparison between WB & SB.

Similarities:
• Electrophoretically separated components
(proteins in WB & DNA in SB), are transferred
from a gel to a solid support and probed with
reagents that are specific for particular
sequences of AA (WB) or nucleotides (SB).
 Comparison between WB & SB, Contnd…

Differences:
The critical difference between SB & WB is: the
nature of the probes

In WB
Probes usually are Ab(s) that
react specifically with Ag-ic
determinants (epitopes)
displayed by the target protein
In SB
NA probes hybridize with
a specificity & rate that
can be predicted by
simple equations,
 Application
1.The confirmatory HIV test

2.Western blot is also used as the definitive test for

Bovine spongiform encephalopathy (BSE(

3.Some forms of Lyme disease testing employ Western

blotting .
 Comparison of Southern, Northern, and
Western blotting techniques
Southern blotting Northern blotting Western blotting
Molecule DNA (ds) mRNA (ss) Protein
detected
Gel Agarose gel Formaldehyde Polyacrylamide gel
electrophoresi agarose gel
s
Gel Depurination, - -
pretreatment denaturation, and
neutralization
Blotting Capillary transfer Capillary transfer Electric transfer
method
Probes DNA cDNA, cRNA primary antibody
Radioactive or Radioactive or
nonradioactive nonradioactive
Detection Autoradiography Autoradiography Chemiluminescent
system Chemiluminescent Chemiluminescent Colorimetric
Colorimetric Colorimetric
 References
• Lippincott, Illustrated review of Biochemistry, 4th
edition
• Molecular Cloning: A Laboratory Manual, J
Sambrook, EF Fritsch, T Maniatis
• Catalogues of some commercial companies
THANK YOU

Dr. Azhar Chishti