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

LECTURE OUTLINES 1. 2. Southern Blotting: 1. History 2. Main use 3. Advantages 4. LECTURE OUTLINES 1. 2. Southern Blotting: 1. History 2. Main use 3. Advantages 4. Probes 5. Hybridization 6. Procedure 7. Steps 8. Methods of Transfer 9. Example of application of SB for the diagnosis of diseases (SCA) Northern Blotting: 1. History 2. Definition 3. Basic steps 4. Applications Dr. Azhar Chishti 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)

OBJECTIVES To understand the basic concept of blotting techniques (Southern, northern, western) To know 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

BLOTTING 1. SOUTHERN BLOT 2. NORTHERN BLOT 3. WESTERN Dr. Azhar Chishti BLOT BLOTTING 1. SOUTHERN BLOT 2. NORTHERN BLOT 3. WESTERN Dr. Azhar Chishti BLOT

Blotting: History Southern Blotting is named after its inventor, the British biologist Edwin Southern 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

SOUTHERN BLOTTING ? Experimental procedure DNA is extracted from cells, leukocytes. DNA is cleaved SOUTHERN BLOTTING ? Experimental procedure DNA is extracted from cells, leukocytes. DNA is cleaved into many fragments by restriction enzyme (Bam. H 1, Eco. R 1 etc) Dr. Azhar Chishti

 The resulting fragments are separated on the basis of size by electrophoresis. The 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

 The DNA fragments are denatured and transferred to nitrocellulose membrane (NYTRAN) for analysis. 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

 DNA segments were visualized by a nonspecific technique, they would appear as an 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

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

Probes • Labeled material to detect a target. • For DNA: 20 -30 nucleotides, 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. 32 P • Sensitive • Relatively cheap • Hazardous You should follow the radioactive waste disposal regulations. Probe * Target DNA • Non-radioactive e. g. Biotin • Sensitive • Relatively expensive Biotin Probe Target DNA Dr. Azhar Chishti * Avidin

Hybridization The binding between ss labeled probe to a complementary nucleotide sequence on the 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

Detection of mutations The presence of a mutation affecting a restriction site causes the 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

Dr. Azhar Chishti Dr. Azhar Chishti

1 - DNA extraction 6 - Detection 2 - DNA cleavage (RE) 3 - 1 - DNA extraction 6 - Detection 2 - DNA cleavage (RE) 3 - DNA Electrophoresis (based on size) 5 Hybridization e. g. with 32 Plabeled probe - + Dr. Azhar Chishti 4 - DNA Denature, Transfer, blocking,

Steps Digestion of genomic DNA (w/ ≥ one RE) DNA fragments Size-separation of the 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) Dr. Azhar Chishti

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

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

weight tight connection DNA eluted from the gel by the moving stream of buffer 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

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

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

Northern Blotting Northern Hybridization A northern blot is a method routinely used in molecular 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

Basis Steps of NB 1. Isolation of intact m. RNA 2. Separation of RNA Basis Steps of NB 1. Isolation of intact m. RNA 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. Dr. Azhar Chishti

Applications Study of gene expression in eukaryotic cells: To measure the amount & size 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

Examples of methods to equalize the amounts of RNA loaded into lanes of gels Examples of methods to equalize the amounts of RNA loaded into lanes of gels OD 260 Use of housekeeping gene (endogenous constitutivelyexpressed gene): Normalizing samples according to their content of m. RNAs of this housekeeping gene Dr. Azhar Chishti

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

WB: Definition Blotting is the transfer of separated proteins from the gel matrix into 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

Applications & Advantages Applications: To determine the molecular weight of a protein (identification) To 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 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

Western blotting The main steps of blotting technique in a chronological order will be 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

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

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

Factors Affecting Transfer Efficiency 1. The Composition of the gel 2. Whethere is complete Factors Affecting Transfer Efficiency 1. The Composition of the gel 2. Whethere is complete contact of the gel with the membrane 3. The position of the electrodes 4. The transfer time 5. The size & composition of proteins 6. The field strength 7. The presence of detergents Dr. Azhar Chishti

WB Procedure; Briefly… 2 1 3 www. bio. davidson. edu/. . . /method/Westernblot. html WB Procedure; Briefly… 2 1 3 www. bio. davidson. edu/. . . /method/Westernblot. html 4 Dr. Azhar Chishti

Direct Detection Method Dr. Azhar Chishti Direct Detection Method Dr. Azhar Chishti

Indirect Detection Method Dr. Azhar Chishti Indirect Detection Method Dr. Azhar Chishti

WB: examples of used substrates Dr. Azhar Chishti WB: examples of used substrates Dr. Azhar Chishti

Chemiluminescent substrates Dr. Azhar Chishti Chemiluminescent substrates Dr. Azhar Chishti

Enhanced Chemi. Fluoresenct (ECF) WB Detection Dr. Azhar Chishti Enhanced Chemi. Fluoresenct (ECF) WB Detection Dr. Azhar Chishti

Western Blotting Procedure; Illustrated Dr. Azhar Chishti Western Blotting Procedure; Illustrated Dr. Azhar Chishti

Steps of WB Dr. Azhar Chishti Steps of WB Dr. Azhar Chishti

Steps of WB Dr. Azhar Chishti Steps of WB Dr. Azhar Chishti

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

Blocking of Blot Several measures should be followed to decrease the nonspecific reactions to 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

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

Primary Antibody labeling The immobilized proteins on the surface of the membrane can be 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 nonradioactive method. Dr. Azhar Chishti

Primary Antibody probing The blot is first incubated with a primary antibody followed by 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 antimouse secondary antibody. Dr. Azhar Chishti

Steps of WB Dr. Azhar Chishti Steps of WB Dr. Azhar Chishti

Steps of WB Dr. Azhar Chishti Steps of WB Dr. Azhar Chishti

Detection and interpretation A prestained MW standard is included in a separate lane during 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

Detection and interpretation (continue) Quantification of a specific protein band can be achieved by 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

Comparison between WB & SB. Similarities: Electrophoretically separated components (proteins in WB & DNA 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

Comparison between WB & SB, Contnd… Differences: The critical difference between SB & WB 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 Dr. Azhar Chishti NA probes hybridize with a specificity & rate that can be predicted by simple equations,

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

THANK YOU Dr. Azhar Chishti THANK YOU Dr. Azhar Chishti