Investigations of infertility Dr. Reem Sallam Endocrinology Block 28 April 2014.
BLOTTING Dr. Reem M. Sallam. OBJECTIVES To understand the basic concept of blotting techniques...
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Transcript of BLOTTING Dr. Reem M. Sallam. OBJECTIVES To understand the basic concept of blotting techniques...
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