BLOTTING Dr. Reem M. Sallam

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)

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)

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

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

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

Probes * * Labeled material to detect a target. For DNA: 20-30 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

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

SB procedure Reem M. Sallam, MD, PhD

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

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 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 Blotting Northern 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 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

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 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 Probe with specific Ab Membrane Reem M. Sallam, MD, PhD

WB Procedure; Briefly… 1 2 4 3 www.bio.davidson.edu/.../method/Westernblot.html Reem M. Sallam, MD, PhD

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

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 WB Why to block? To increase sensitivity To prevent nonspecific signal Reem M. Sallam, MD, PhD

For Direct Transfer, choices are: 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 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

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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