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BLOTTING

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

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BLOTTING

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  1. 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: • History • 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

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

  5. 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

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

  7. 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

  8. 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

  9. 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

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

  11. * • Avidin Biotin • * Probe Probe • Target DNA • Target DNA 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 Dr. Azhar Chishti

  12. 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

  13. 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

  14. Dr. Azhar Chishti

  15. 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,

  16. 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. AzharChishti

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

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

  19. 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

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

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

  22. Northern BlottingNorthern 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

  23. 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

  24. 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

  25. Examples of methods to equalize the amounts of RNA loaded into lanes of gels • OD260 • Use of housekeeping gene (endogenous constitutively-expressed gene): Normalizing samples according to their content of mRNAs of this housekeeping gene Dr. Azhar Chishti

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

  27. 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 NatlAcadSci U S A. 76 (9): 4350–4354 Dr. Azhar Chishti

  28. 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. AzharChishti

  29. 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

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

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

  32. 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

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

  34. Direct Detection Method Dr. Azhar Chishti

  35. Indirect Detection Method Dr. Azhar Chishti

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

  37. Chemiluminescent substrates Dr. Azhar Chishti

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

  39. Western Blotting Procedure; Illustrated Dr. Azhar Chishti

  40. Steps of WB Dr. Azhar Chishti

  41. Steps of WB Dr. Azhar Chishti

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

  43. 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

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

  45. 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

  46. 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. AzharChishti

  47. Steps of WB Dr. Azhar Chishti

  48. Steps of WB Dr. Azhar Chishti

  49. 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. AzharChishti

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