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Dr. Azhar ChishtiDepartment of Medical
Biochemistry
Dr. Azhar Chishti
Dr. Azhar Chishti
LECTURE OUTLINES1. Southern Blotting:
1. History2. Main use3. Advantages4. Probes5. Hybridization6. Procedure7. Steps8. Methods of Transfer9. Example of application of
SB for the diagnosis of diseases (SCA)
2. Northern Blotting:1. History2. Definition3. Basic steps4. Applications
3. Western Blotting:1. WB: Definition2. Applications &
Advantages3. WB: An overview4. Direction of transfer5. Factors Affecting
Transfer Efficiency6. WB procedure, briefly7. WB Detection methods8. Examples of used
substrates9. WB procedure,
illustrated10. Comparison between SB
& WB (Similarities & Differences)
Dr. Azhar Chishti
OBJECTIVESTo 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)
1. SOUTHERN BLOT
2. NORTHERN BLOT
3. WESTERN BLOT
Dr. Azhar Chishti
Dr. Azhar Chishti
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.
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
Dr. Azhar Chishti
•Labeled material to detect a target.
•For DNA: 20-30 nucleotides, complementary to a region in the gene
•Methods of labeling: •Non-radioactive e.g. Biotin•Radioactive e.g. 32P
•Sensitive•Relatively cheap•HazardousYou should follow the radioactive waste disposal regulations.
•Sensitive•Relatively expensive
Target DNA
ProbeBiotin Avidin*
Target DNA
Probe *
Probes
Dr. Azhar Chishti
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.
Hybridization
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
Dr. Azhar Chishti
1- DNA extraction
2- DNA cleavage (RE)
3- DNA Electrophoresis (based on size) -
+
4- DNA Denature, Transfer, blocking,
5- Hybridization e.g. with 32P-labeled probe
6- Detection
Dr. Azhar Chishti
StepsDigestion 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)
METHODS OF TRANSFER
Downward Capillary Transfer
Upward Capillary Transfer
Simultaneous Transfer to Two Membranes
Electrophoretic Transfer
Vacuum Transfer
Dr. Azhar Chishti
Example of TransferUpward Capillary Transfer
Weight
Glass Plate
Whatman 3MM paper
Gel
Paper towels
Membrane (nylon or nitrocellulose)
Whatman 3MM paper
Transfer buffer
Dr. Azhar Chishti
Buffer drawn from a reservoir passes through the gel into a stack of paper towels
DNA eluted from the gel by the moving stream of buffer is deposited onto a membrane
weight tight connection
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 BlottingNorthern 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 NB1. 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.
ApplicationsStudy 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 gelsDr. Azhar Chishti
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 Towbin H, et al (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.". sheets: procedure and some applications.". Proc Natl Proc Natl Acad Sci U S A.Acad Sci U S A. 76 (9): 4350–4354 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 techniqueAs little as 1-5 ng of an average-sized protein can be detected by WB
Western blotting
The main steps of blotting technique in a chronological order will be as follows:
BlockingProbing with the specific antibody(ies)WashDetection WashingX-ray (Gel Documentation System)
Dr. Azhar Chishti
Dr. Azhar Chishti
Electrophoretic Transfer: An Overview
Important Issue:Important Issue:
Where to put the gel and the membrane relative to Where to put the gel and the membrane relative to the electroblotting transfer electrodes?the electroblotting transfer electrodes?
Dr. Azhar Chishti
Direction of Transfer
Perpendicularly from the direction of travel of proteins through the separating gel
Gel
Membrane
Probe with specific Ab
Dr. Azhar Chishti
Factors Affecting Transfer Efficiency
1. The Composition of the gel2. Whether there is complete
contact of the gel with the membrane
3. The position of the electrodes4. The transfer time5. The size & composition of
proteins6. The field strength7. The presence of detergents
Dr. Azhar Chishti
WB Procedure; Briefly…
www.bio.davidson.edu/.../method/Westernblot.html
12
3 4
Dr. Azhar Chishti
Direct Detection Method
Dr. Azhar Chishti
Indirect Detection Method
Dr. Azhar Chishti
WB: examples of used substrates
Dr. Azhar Chishti
Dr. Azhar Chishti
Dr. Azhar Chishti
Dr. Azhar Chishti
Dr. Azhar Chishti
Why to block?Why to block?To increase sensitivityTo increase sensitivityTo prevent nonspecific signalTo prevent nonspecific signal
Dr. Azhar Chishti
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
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.
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 anti- mouse secondary antibody.
Dr. Azhar Chishti
Dr. Azhar Chishti
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
Probes usually are Ab(s) that react specifically with Ag-ic determinants (epitopes) displayed by the target protein
NA probes hybridize with a specificity & rate that can be predicted by simple equations,
In WB In SB
Dr. Azhar Chishti
ReferencesLippincott, Illustrated review of
Biochemistry, 4th editionMolecular Cloning: A Laboratory Manual,
J Sambrook, EF Fritsch, T Maniatis Catalogues of some commercial
companies
Dr. Azhar Chishti
Dr. Azhar Chishti
THANK YOU