BLOTTINGDr. Reem M. Sallam

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)

LECTURE OUTLINES1. Southern Blotting:

1. History2. Main use3. Advantages4. Probes5. Hybridization6. Procedure7. Steps8. 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)

Southern Blotting“Southern Hybridization”

Reem M. Sallam, MD, PhD

SB: Definition

A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples.

It combines: transfer of electrophoresis-separated DNA

fragments to a filter membrane subsequent fragment detection by probe

hybridization.

Reem M. Sallam, MD, PhD

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.

Reem M. Sallam, MD, PhD

Is used to study how genes are organized within genomes by mapping restriction sites in & around segments of genomic DNA for which specific probes are available It can detect mutations in DNA

It combines the use of RE, electrophoresis, DNA probes

A mutation may: alter the restriction recognition site RE fails to

recognize & cleave that site or create a new cleavage site new restriction

fragments. Or may be revealed by using a different RE.

SB: Main use

Reem M. Sallam, MD, PhD

SB: Advantages

Nowadays, immaculate results are the general rule due to the significant improvement of the sensitivity & reproducibility of the technique

Reem M. Sallam, MD, PhD

• 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

Reem M. Sallam, MD, PhD

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

Reem M. Sallam, MD, PhD

SB procedure

Reem M. Sallam, MD, PhD

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

Reem M. Sallam, MD, PhD

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)

Reem M. Sallam, MD, PhD

Example of Application of SB in diagnosis of mutation in globin gene

Reem M. Sallam, MD, PhD

Reem M. Sallam, MD, PhD

Northern BlottingNorthern Hybridization

Reem M. Sallam, MD, PhD

History

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

Reem M. Sallam, MD, PhD

NB: Definition

A northern blot is a method routinely used in molecular biology for detection of a specific RNA sequence in RNA samples.

Reem M. Sallam, MD, PhD

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.

Reem M. Sallam, MD, PhD

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

Reem M. Sallam, MD, PhD

Western Blotting“Immunoblotting”= electrophoretic transfer of

proteins from gels to membranes

Reem M. Sallam, MD, PhD

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

Reem M. Sallam, MD, PhD

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.

Advantages:WB is highly sensitive technique

As little as 1-5 ng of an average-sized protein can be detected by WB

Reem M. Sallam, MD, PhD

Electrophoretic Transfer: An Overview

Important Issue:Where to put the gel and the membrane relative to

the electroblotting transfer electrodes? Reem M. Sallam, MD, PhD

Direction of Transfer

Perpendicularly from the direction of travel of proteins through the separating gel

Gel

Membrane

Probe with specific Ab

Reem M. Sallam, MD, PhD

WB Procedure; Briefly…

www.bio.davidson.edu/.../method/Westernblot.html

12

3 4

Reem M. Sallam, MD, PhD

WB Detection Methods

Radioactive Non-Radioactive

FluorescenceSensitiveSafe (non-radioactive)QuantitativeFaster than ChemiluminescenceStable signal

Chemiluminescence e.g. ECL

SensitiveSafe (non-radioactive)Quantitative

Reem M. Sallam, MD, PhD

Detection Methods

Reem M. Sallam, MD, PhD

Direct Detection Method

Reem M. Sallam, MD, PhD

Indirect Detection Method

Reem M. Sallam, MD, PhD

WB: examples of used substrates

Reem M. Sallam, MD, PhD

Chemiluminescent substrates

Reem M. Sallam, MD, PhD

Enhanced ChemiFluoresenct (ECF) WB Detection

Reem M. Sallam, MD, PhD

Western Blotting Procedure; Illustrated

Reem M. Sallam, MD, PhD

Steps of WB

Reem M. Sallam, MD, PhD

Steps of WB

Reem M. Sallam, MD, PhD

Steps of WBWhy to block?

To increase sensitivityTo prevent nonspecific signal

Reem M. Sallam, MD, PhD

Steps of WB

For Direct Transfer, choices are:

Reem M. Sallam, MD, PhD

Steps of WB

Reem M. Sallam, MD, PhD

Steps of WB

Reem M. Sallam, MD, PhD

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).

Reem M. Sallam, MD, PhD

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

Reem M. Sallam, MD, PhD

References

Lippincott, Illustrated review of Biochemistry, 4th edition

Molecular Cloning: A Laboratory Manual, J Sambrook, EF Fritsch, T Maniatis

Catalogues of some commercial companies

Reem M. Sallam, MD, PhD

THANK YOU