Transcript BLOTTING.142

Dr. Azhar Chishti
Department of Medical Biochemistry
Dr. Azhar Chishti
LECTURE OUTLINES
1.
2.
Southern Blotting:
1.
History
2.
Main use
3.
Advantages
4.
Probes
5.
Hybridization
6.
Procedure
7.
Steps
8.
Methods of Transfer
9.
Example of application of SB
for the diagnosis of diseases
(SCA)
Northern Blotting:
1.
History
2.
Definition
3.
Basic steps
4.
Applications
Dr. Azhar Chishti
3.
Western Blotting:
1.
WB: Definition
2.
Applications & Advantages
3.
WB: An overview
4.
Direction of transfer
5.
Factors Affecting Transfer
Efficiency
6.
WB procedure, briefly
7.
WB Detection methods
8.
Examples of used substrates
9.
WB procedure, illustrated
10. Comparison between SB &
WB (Similarities &
Differences)
OBJECTIVES
 To understand the basic concept of blotting
techniques (Southern, northern, western)
 To know the main applications and advantages of each
of the main types of blotting techniques
 To be familiar with the steps (in brief) for performing
a blotting procedure
 To understand the major similarities & differences
between different blotting techniques
 To be introduced to an example of applying a blotting
technique in diagnosis of diseases (SCA)
Dr. Azhar Chishti
1. SOUTHERN BLOT
2. NORTHERN BLOT
3. WESTERN
Dr. Azhar Chishti
BLOT
Blotting: History
 Southern Blotting is named after its
inventor, the British biologist Edwin
Southern (1975)
 Other blotting methods (i.e. western
blot, WB, northern blot, NB) that
employ similar principles, but using
protein or RNA, have later been named
in reference to Edwin Southern's name.
Dr. Azhar Chishti
SOUTHERN BLOTTING ?
Experimental procedure
 DNA is extracted from cells, leukocytes.
 DNA is cleaved into• many fragments by
restriction enzyme (BamH1, EcoR1 etc)
Dr. Azhar Chishti
 The resulting fragments are separated on
the basis of size by electrophoresis.
 The large fragments move more slowly than
the smaller fragments.
 The lengths of the fragments are compared
with band of relative standard fragments of
known size.
Dr. Azhar Chishti
 The DNA fragments are denatured and
transferred to nitrocellulose membrane
(NYTRAN) for analysis.
 DNA represents the individual's entire
genome, the enzymic digest contains a
million or more fragments.
 The gene of interest is on only one of these
pieces of DNA.
Dr. Azhar Chishti
 DNA segments were visualized by a
nonspecific technique, they would appear as
an unresolved blur of overlapping bands.
 To avoid this, the last step in Southern
blotting uses a probe to identify the DNA
fragments of interest.
Dr. Azhar Chishti
 Southern blot analysis depend on the
specific restriction endonuclease
 The probe used to visualize the restriction
fragments.
Dr. Azhar Chishti
Probes
•Labeled material to detect a target.
•For DNA: 20-30 nucleotides, complementary to a
region in the gene
•Methods of labeling:
•Radioactive e.g. 32P
•Sensitive
•Relatively cheap
•Hazardous
You should follow the
radioactive waste disposal
regulations.
Probe
*
Target DNA
•Non-radioactive e.g. Biotin
•Sensitive
•Relatively expensive
Biotin
Probe
Target DNA
Dr. Azhar Chishti
*
Avidin
Hybridization
The binding between ss labeled probe to a
complementary nucleotide sequence on the target DNA.
Degree of hybridization depends on method of probe
labeling (radioacitve or non-radioactive system e.g.
biotin-avidin.
Dr. Azhar Chishti
Detection of mutations
 The presence of a mutation affecting a
restriction site causes the pattern of bands to
differ from those seen with a normal gene.
 A change in one nucleotide may alter the
nucleotide sequence so that the restriction
endonuclease fails to recognize and cleave at
that site
(for example, in Figure, person 2 lacks a
restriction site present in person 1).
Dr. Azhar Chishti
Dr. Azhar Chishti
1- DNA extraction
6- Detection
2- DNA cleavage
(RE)
3- DNA
Electrophoresis
(based on size)
5Hybridization
e.g. with 32Plabeled probe
-
+
DNA
Dr. Azhar4Chishti
Denature, Transfer, blocking,
Steps
 Digestion of genomic DNA (w/ ≥ one RE)  DNA fragments
 Size-separation of the fragments (standard agarose gel electrophoresis)
 In situ denaturation of the DNA fragments (by incubation @ ↑temp)
 Transfer of denatured DNA fragments into a solid support (nylon or
nitrocellulose).
 Hybridization of the immobilized DNA to a labeled probe (DNA, RNA)
 Detection of the bands complementary to the probe (e.g. by
autoradiography)
 Estimation of the size & number of the bands generated after digestion
of the genomic DNA w/ different RE  placing the target DNA within a
context of restriction sites)
Dr. Azhar Chishti
METHODS OF TRANSFER
Upward Capillary Transfer
Downward Capillary Transfer
Simultaneous Transfer to Two Membranes
Electrophoretic Transfer
Vacuum Transfer
Example of Transfer
Upward Capillary Transfer
Weight
Glass Plate
Paper towels
Whatman 3MM paper
Membrane (nylon
or nitrocellulose)
Whatman 3MM
paper
Gel
Transfer buffer
Dr. Azhar Chishti
weight  tight connection
DNA eluted from
the gel by the
moving stream of
buffer is
deposited onto a
membrane
Buffer drawn from
a reservoir passes
through the gel
into a stack of
paper towels
Dr. Azhar Chishti
Example of Application of SB in diagnosis
of mutation in  globin gene
Dr. Azhar Chishti
Example of Application of SB in diagnosis
of mutation in  globin gene
Dr. Azhar Chishti
Northern Blotting
Northern Hybridization
A northern blot is a method routinely used in
molecular biology for detection of a specific RNA
sequence in RNA samples.
 The method was first described in the seventies
(Alwine et al. 1977, 1979)
 It is still being improved (Kroczek 1993), with the
basic steps remaining the same
Dr. Azhar Chishti
Basis Steps of NB
1. Isolation of intact mRNA
2. Separation of RNA according to size (through a
denaturing agarose gel e.g. with Glyoxal/formamide)
Transfer of the RNA to a solid support
Fixation of the RNA to the solid matrix
Hybridization of the immobilized RNA to probes
complementary to the sequences of interest
Removal of probe molecules that are nonspecifically
bound to the solid matrix
Detection, capture, & analysis of an image of the
specifically bound probe molecules.
Dr. Azhar Chishti
Applications
 Study of gene expression in eukaryotic cells:
 To measure the amount & size of RNAs
transcribed from eukaryotic genes
 To estimate the abundance of RNAs
 Therefore, it is crucially important to
equalize the amounts of RNA loaded into
lanes of gels
Dr. Azhar Chishti
Examples of methods to equalize the amounts of
RNA loaded into lanes of gels
 OD260
 Use of housekeeping gene (endogenous constitutively-
expressed gene): Normalizing samples according to
their content of mRNAs of this housekeeping gene
Dr. Azhar Chishti
Western Blotting
“Immunoblotting”
= electrophoretic transfer of proteins
from gels to membranes
Dr. Azhar Chishti
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
Dr. Azhar Chishti
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
Dr. Azhar Chishti
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)
Dr. Azhar Chishti
Electrophoretic Transfer: An Overview
Important Issue:
Where to put the gel and the membrane relative to
the electroblotting transfer electrodes?
Dr. Azhar Chishti
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
3.
4.
5.
6.
7.
the gel with the membrane
The position of the electrodes
The transfer time
The size & composition of proteins
The field strength
The presence of detergents
Dr. Azhar Chishti
WB Procedure; Briefly…
2
1
3
www.bio.davidson.edu/.../method/Westernblot.html
4
Dr. Azhar Chishti
Direct Detection Method
Dr. Azhar Chishti
Indirect Detection Method
Dr. Azhar Chishti
WB: examples of used
substrates
Dr. Azhar Chishti
Chemiluminescent substrates
Dr. Azhar Chishti
Enhanced ChemiFluoresenct (ECF) WB
Detection
Dr. Azhar Chishti
Dr. Azhar Chishti
Steps of WB
Dr. Azhar Chishti
Steps of WB
Dr. Azhar Chishti
Why to block?
To increase sensitivity
To prevent nonspecific signal
Dr. Azhar Chishti
Steps of WB
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.
Dr. Azhar Chishti
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 nonradioactive method.
Dr. Azhar Chishti
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 antimouse secondary antibody.
Dr. Azhar Chishti
Steps of WB
Dr. Azhar Chishti
Steps of WB
Dr. Azhar Chishti
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.
Dr. Azhar Chishti
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.
Dr. Azhar Chishti
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).
Dr. Azhar Chishti
Comparison between WB & SB,
Contnd…
Differences:
The critical difference between SB & WB is: the nature
of the probes
In WB
In SB
Probes usually are Ab(s) that react
specifically with Ag-ic determinants
(epitopes) displayed by the target
protein
Dr. Azhar Chishti
NA probes hybridize with a
specificity & rate that can be
predicted by simple equations,
References
 Lippincott, Illustrated review of Biochemistry, 4th
edition
 Molecular Cloning: A Laboratory Manual, J
Sambrook, EF Fritsch, T Maniatis
 Catalogues of some commercial companies
Dr. Azhar Chishti
THANK YOU
Dr. Azhar Chishti