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 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)

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

5. 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

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. 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

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. 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

10. 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

11. SB procedure
Reem M. Sallam, MD, PhD

12. 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

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. Reem M. Sallam, MD, PhD

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 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 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

20. 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

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
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

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

26. WB Procedure; Briefly…1 2 4 3
Reem M. Sallam, MD, PhD

27. 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

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. For Direct Transfer, choices are:
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
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

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 