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BLOTTING Dr. Reem M. Sallam. OBJECTIVES To understand the basic concept of blotting techniques (Southern, northern, western) To know the main applications.

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Presentation on theme: "BLOTTING Dr. Reem M. Sallam. OBJECTIVES To understand the basic concept of blotting techniques (Southern, northern, western) To know the main applications."— Presentation transcript:

1 BLOTTING Dr. Reem M. Sallam

2 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)

3 LECTURE OUTLINES 1. 1.Southern Blotting: 1. History 2. Main use 3. Advantages 4. Probes 5. Hybridization 6. Procedure 7. Steps 8. Example of application of SB for the diagnosis of diseases (SCA) 2. 2.Northern Blotting: 1. History 2. Definition 3. Basic steps 4. Applications 3. 3.Western Blotting: 1. WB: Definition 2. Applications & Advantages 3. WB: An overview 4. Direction of transfer 5. Factors Affecting Transfer Efficiency 6. WB procedure, briefly 7. WB Detection methods 8. Examples of used substrates 9. WB procedure, illustrated 10. Comparison between SB & WB (Similarities & Differences)

4 Southern Blotting Southern Hybridization Reem M. Sallam, MD, PhD

5 SB: Definition A is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. 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 transfer of electrophoresis-separated DNA fragments to a filter membrane subsequent fragment detection by probe hybridization. subsequent fragment detection by probe hybridization. Reem M. Sallam, MD, PhD

6 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

7 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 alter the restriction recognition site RE fails to recognize & cleave that site or create a new cleavage site new restriction fragments. or create a new cleavage site new restriction fragments. Or may be revealed by using a different RE. Or may be revealed by using a different RE. SB: Main use Reem M. Sallam, MD, PhD

8 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

9 Labeled material to detect a target. For DNA: nucleotides, complementary to a region in the gene Methods of labeling: Non-radioactive e.g. Biotin Radioactive e.g. 32 P Sensitive Relatively cheap Hazardous You should follow the radioactive waste disposal regulations. Sensitive Relatively expensive Target DNA Probe Biotin Avidin * Target DNA Probe * Probes Reem M. Sallam, MD, PhD

10 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

11 SB procedure Reem M. Sallam, MD, PhD

12 1- DNA extraction 2- DNA cleavage (RE) 3- DNA Electrophoresis (based on size) DNA Denature, Transfer, blocking, 5- Hybridization e.g. with 32 P- labeled probe 6- Detection Reem M. Sallam, MD, PhD

13 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

14 Example of Application of SB in diagnosis of mutation in globin gene Reem M. Sallam, MD, PhD

15

16 Northern Blotting Northern Hybridization Reem M. Sallam, MD, PhD

17 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

18 NB: Definition A is a method routinely used in molecular biology for detection of a specific RNA sequence in RNA samples. 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

19 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

20 Applications Study of gene expression in eukaryotic cells: To & of RNAs transcribed from eukaryotic genes To measure the amount & size of RNAs transcribed from eukaryotic genes To estimate the of RNAs To estimate the abundance of RNAs Therefore, it is crucially important to loaded into lanes of gels Therefore, it is crucially important to equalize the amounts of RNA loaded into lanes of gels Reem M. Sallam, MD, PhD

21 Western Blotting Immunoblotting = electrophoretic transfer of proteins from gels to membranes Reem M. Sallam, MD, PhD

22 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

23 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 of an average-sized protein can be detected by WB As little as 1-5 ng of an average-sized protein can be detected by WB Reem M. Sallam, MD, PhD

24 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

25 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

26 WB Procedure; Briefly… Reem M. Sallam, MD, PhD

27 WB Detection Methods Radioactive Non-Radioactive Fluorescence Sensitive Safe (non-radioactive) Quantitative Faster than Chemiluminescence Stable signal Chemiluminescence e.g. ECL Sensitive Safe (non-radioactive) Quantitative Reem M. Sallam, MD, PhD

28 Detection Methods Reem M. Sallam, MD, PhD

29 Direct Detection Method Reem M. Sallam, MD, PhD

30 Indirect Detection Method Reem M. Sallam, MD, PhD

31 WB: examples of used substrates Reem M. Sallam, MD, PhD

32 Chemiluminescent substrates Reem M. Sallam, MD, PhD

33 Enhanced ChemiFluoresenct (ECF) WB Detection Reem M. Sallam, MD, PhD

34 Western Blotting Procedure; Illustrated Reem M. Sallam, MD, PhD

35 Steps of WB Reem M. Sallam, MD, PhD

36 Steps of WB Reem M. Sallam, MD, PhD

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

38 Steps of WB For Direct Transfer, choices are: Reem M. Sallam, MD, PhD

39 Steps of WB Reem M. Sallam, MD, PhD

40 Steps of WB Reem M. Sallam, MD, PhD

41 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

42 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

43 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

44 THANK YOU THANK YOU


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