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BLOTTING Dr. Reem M. Sallam
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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)
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LECTURE OUTLINES Southern Blotting: History Main use Advantages Probes
Hybridization Procedure Steps Example of application of SB for the diagnosis of diseases (SCA) Northern Blotting: Definition Basic steps Applications Western Blotting: WB: Definition Applications & Advantages WB: An overview Direction of transfer Factors Affecting Transfer Efficiency WB procedure, briefly WB Detection methods Examples of used substrates WB procedure, illustrated Comparison between SB & WB (Similarities & Differences)
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Southern Blotting “Southern Hybridization”
Reem M. Sallam, MD, PhD
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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
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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
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SB: Main use 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. Reem M. Sallam, MD, PhD
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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
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Probes * * Labeled material to detect a target.
For DNA: nucleotides, complementary to a region in the gene Methods of labeling: Radioactive e.g. 32P Non-radioactive e.g. Biotin Sensitive Relatively cheap Hazardous You should follow the radioactive waste disposal regulations. Sensitive Relatively expensive Target DNA Probe Biotin Avidin * Target DNA Probe * Reem M. Sallam, MD, PhD
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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. Reem M. Sallam, MD, PhD
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SB procedure Reem M. Sallam, MD, PhD
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4- DNA Denature, Transfer, blocking,
1- DNA extraction 6- Detection 2- DNA cleavage (RE) 5- Hybridization e.g. with 32P-labeled probe 3- DNA Electrophoresis (based on size) - + 4- DNA Denature, Transfer, blocking, Reem M. Sallam, MD, PhD
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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 ↑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
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Example of Application of SB in diagnosis of mutation in globin gene
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Reem M. Sallam, MD, PhD
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Northern Blotting Northern Hybridization
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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
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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
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Basis Steps of NB Isolation of intact mRNA
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
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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
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Western Blotting “Immunoblotting”
= electrophoretic transfer of proteins from gels to membranes Reem M. Sallam, MD, PhD
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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
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Applications & Advantages
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
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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
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Direction of Transfer Perpendicularly from the direction of travel of proteins through the separating gel Gel Probe with specific Ab Membrane Reem M. Sallam, MD, PhD
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WB Procedure; Briefly…
1 2 4 3 Reem M. Sallam, MD, PhD
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Chemiluminescence e.g. ECL
WB Detection Methods Radioactive Non-Radioactive Chemiluminescence e.g. ECL Sensitive Safe (non-radioactive) Quantitative Fluorescence Sensitive Safe (non-radioactive) Quantitative Faster than Chemiluminescence Stable signal Reem M. Sallam, MD, PhD
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Detection Methods Reem M. Sallam, MD, PhD
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Direct Detection Method
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Indirect Detection Method
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WB: examples of used substrates
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Chemiluminescent substrates
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Enhanced ChemiFluoresenct (ECF) WB Detection
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Western Blotting Procedure; Illustrated
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Steps of WB Reem M. Sallam, MD, PhD
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Steps of WB Reem M. Sallam, MD, PhD
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Steps of WB Why to block? To increase sensitivity
To prevent nonspecific signal Reem M. Sallam, MD, PhD
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For Direct Transfer, choices are:
Steps of WB For Direct Transfer, choices are: Reem M. Sallam, MD, PhD
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Steps of WB Reem M. Sallam, MD, PhD
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Steps of WB Reem M. Sallam, MD, PhD
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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
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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 NA probes hybridize with a specificity & rate that can be predicted by simple equations, Reem M. Sallam, MD, PhD
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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
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THANK YOU
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