Dr. Azhar Chishti Department of Medical Biochemistry Dr. Azhar Chishti.

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