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Dr. Azhar Chishti Department of Medical Biochemistry

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1 Dr. Azhar Chishti Department of Medical Biochemistry
BLOTTING Dr. Azhar Chishti Department of Medical Biochemistry Dr. Azhar Chishti

2 LECTURE OUTLINES Southern Blotting: History Main use Advantages Probes
Hybridization Procedure Steps Methods of Transfer 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) Dr. Azhar Chishti

3 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) Dr. Azhar Chishti


Dr. Azhar Chishti

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. Dr. Azhar Chishti

7 SOUTHERN BLOTTING ? DNA is extracted from cells, leukocytes.
Experimental procedure DNA is extracted from cells, leukocytes. DNA is cleaved into many fragments by restriction enzyme (BamH1, EcoR1 etc) Dr. Azhar Chishti

8 The large fragments move more slowly than the smaller fragments.
The resulting fragments are separated on the basis of size by electrophoresis. The large fragments move more slowly than the smaller fragments. The lengths of the fragments are compared with band of relative standard fragments of known size. Dr. Azhar Chishti

9 The gene of interest is on only one of these pieces of DNA.
The DNA fragments are denatured and transferred to nitrocellulose membrane (NYTRAN) for analysis. DNA represents the individual's entire genome, the enzymic digest contains a million or more fragments. The gene of interest is on only one of these pieces of DNA. Dr. Azhar Chishti

10 DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest. Dr. Azhar Chishti

11 Southern blot analysis depend on the specific restriction endonuclease
The probe used to visualize the restriction fragments. Dr. Azhar Chishti

12 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 * Dr. Azhar Chishti

13 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. Dr. Azhar Chishti

14 Detection of mutations
The presence of a mutation affecting a restriction site causes the pattern of bands to differ from those seen with a normal gene. A change in one nucleotide may alter the nucleotide sequence so that the restriction endonuclease fails to recognize and cleave at that site (for example, in Figure, person 2 lacks a restriction site present in person 1). Dr. Azhar Chishti

15 Dr. Azhar Chishti

16 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) - + Dr. Azhar Chishti 4- DNA Denature, Transfer, blocking,

17 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) Dr. Azhar Chishti

18 METHODS OF TRANSFER Upward Capillary Transfer
Downward Capillary Transfer Simultaneous Transfer to Two Membranes Electrophoretic Transfer Vacuum Transfer

19 Example of Transfer Upward Capillary Transfer
Weight Glass Plate Paper towels Whatman 3MM paper Membrane (nylon or nitrocellulose) Whatman 3MM paper Gel Transfer buffer Dr. Azhar Chishti

20 weight  tight connection
DNA eluted from the gel by the moving stream of buffer is deposited onto a membrane Buffer drawn from a reservoir passes through the gel into a stack of paper towels Dr. Azhar Chishti

21 Example of Application of SB in diagnosis of mutation in  globin gene
Dr. Azhar Chishti

22 Example of Application of SB in diagnosis of mutation in  globin gene
Dr. Azhar Chishti

23 Northern Blotting Northern Hybridization
A northern blot is a method routinely used in molecular biology for detection of a specific RNA sequence in RNA samples. 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 Dr. Azhar Chishti

24 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. Dr. Azhar Chishti

25 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 Dr. Azhar Chishti

26 Examples of methods to equalize the amounts of RNA loaded into lanes of gels
Use of housekeeping gene (endogenous constitutively-expressed gene): Normalizing samples according to their content of mRNAs of this housekeeping gene Dr. Azhar Chishti

27 Western Blotting “Immunoblotting”
= electrophoretic transfer of proteins from gels to membranes Dr. Azhar Chishti

28 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 Dr. Azhar Chishti

29 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 that are not radiolabeled (unlike immunoprecipitation) Advantages: WB is highly sensitive technique As little as 1-5 ng of an average-sized protein can be detected by WB Dr. Azhar Chishti

30 X-ray (Gel Documentation System)
Western blotting The main steps of blotting technique in a chronological order will be as follows: Blocking Probing with the specific antibody(ies) Wash Detection Washing X-ray (Gel Documentation System) Dr. Azhar Chishti

31 Electrophoretic Transfer: An Overview
Important Issue: Where to put the gel and the membrane relative to the electroblotting transfer electrodes? Dr. Azhar Chishti

32 Direction of Transfer Perpendicularly from the direction of travel of proteins through the separating gel Gel Probe with specific Ab Membrane Dr. Azhar Chishti

33 Factors Affecting Transfer Efficiency
The Composition of the gel Whether there is complete contact of the gel with the membrane The position of the electrodes The transfer time The size & composition of proteins The field strength The presence of detergents Dr. Azhar Chishti

34 WB Procedure; Briefly…
1 2 4 3 Dr. Azhar Chishti

35 Direct Detection Method
Dr. Azhar Chishti

36 Indirect Detection Method
Dr. Azhar Chishti

37 WB: examples of used substrates
Dr. Azhar Chishti

38 Chemiluminescent substrates
Dr. Azhar Chishti

39 Enhanced ChemiFluoresenct (ECF) WB Detection
Dr. Azhar Chishti

40 Western Blotting Procedure; Illustrated
Dr. Azhar Chishti

41 Steps of WB Dr. Azhar Chishti

42 Steps of WB Dr. Azhar Chishti

43 Steps of WB Why to block? To increase sensitivity
To prevent nonspecific signal Dr. Azhar Chishti

44 Blocking of Blot Several measures should be followed to decrease the nonspecific reactions to a minimum, i.e., increasing the signal to noise ratio. Blocking step is the incubation of the membrane with solution containing BSA or fat-free milk or casein for a sufficient time with shaking. Dr. Azhar Chishti

45 For Direct Transfer, choices are:
Steps of WB For Direct Transfer, choices are: Dr. Azhar Chishti

46 Primary Antibody labeling
The immobilized proteins on the surface of the membrane can be detected using a specific, labeled antibody. Labeling of the antibody can be performed using a radioactive or non- radioactive method. Dr. Azhar Chishti

47 Primary Antibody probing
The blot is first incubated with a primary antibody followed by the addition of a labeled secondary antibody that has species specificity for the primary one. For example, probing of the membrane using mouse primary antibody and anti- mouse secondary antibody. Dr. Azhar Chishti

48 Steps of WB Dr. Azhar Chishti

49 Steps of WB Dr. Azhar Chishti

50 Detection and interpretation
A prestained MW standard is included in a separate lane during electrophoresis to allow the identification of the MW of the target protein. Similar to the analysis of electrophoresis results on a gel, the data on the membrane can be quantitatively analyzed using gel documentation system. Dr. Azhar Chishti

51 Detection and interpretation (continue)
Quantification of a specific protein band can be achieved by densitometry and integrating the areas under the peaks. Several gel documentation systems are commercially available that can be useful for analysis of results from the gel or membranes. Dr. Azhar Chishti

52 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). Dr. Azhar Chishti

53 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, Dr. Azhar Chishti

54 References Lippincott, Illustrated review of Biochemistry, 4th edition
Molecular Cloning: A Laboratory Manual, J Sambrook, EF Fritsch, T Maniatis Catalogues of some commercial companies Dr. Azhar Chishti

55 THANK YOU Dr. Azhar Chishti

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