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Southern northern and western blotting
Comparison of Southern,
Northern, and Western analyses
of Gene X
Southern hybridization

 First described by E. M. Southern in 1975.
 Applications of Southern hybridization
   RFLP’s, VNTR’s and DNA fingerprinting
   Checking of the gene knockout mice
 The flow chart of Southern hybridization
Southern hybridization




    Transfer buffer
Detection of an RFLP by
Southern blotting
Detection of the sickle-cell
globin gene by Southern
blotting
Checking of the gene knockout
mice
Flow chart of Southern
hybridization
     Preparing the samples and running the gel

                   Southern transfer

                  Probe preparation    Isotope
                                       Non-isotope
                   Prehybridization

                     Hybridization

              Post-hybridization washing

                   Signal detection
Preparing the samples and
running the gel
   Digest 10 pg to 10 µg of desired DNA samples
    to completion.
   Prepare an agarose gel, load samples
    (remember marker), and electrophorese.
   Stain gel ethidium bromide solution (0.5
    µg/ml).
   Photograph gel (with ruler).
Critical parameters (I)
 Note the complexity of DNA
   Genomic DNA
     A single-copy of mammalian gene, 3 Kb
      average in length
     10 µg x 3 Kb/3 x 106 Kb = 10 µg x 1/106 = 10
      pg
   Plasmid DNA or PCR products
      0.1 µg of a 3 Kb plasmid DNA ≅100 ng
Gel treatment

 Acid treatment
   0.2 N HCl solution
 Denaturation
   NaOH solution
 Neutralization
   Tris-Cl buffer (pH8.0)
Southern transfer
            Measure gel and set up transfer
             assembly:
                Wick in tray with 20x SSC
                Gel
                Nitrocellulose or Nylon filters (soaked
                 in H2O and 20x SSC)
                3MM Whatman filter paper
                Paper towels
                Weight
After Southern transfer


            Dissemble transfer
             pyramid and rinse
             nitrocellulose in 2x SSC
            Bake nitrocellulose at 80°C
             for 2 hr or UV-crosslink
             Nylon membrane for
             seconds
Preparation of probes

  Synthesis of uniformly labeled
   double-stranded DNA probes
  Preparation of single-stranded probes
  Labeling the 5′ and 3′ termini of DNA
Synthesis of double-stranded DNA
probes
 - Nick translation of DNA
 - Labeled DNA probes using random
  oligonucleotide primers
Nick translation
Preparation of single-stranded
probes
  Synthesis of single-stranded DNA probes
   using bacteriophage M13 vectors.

  Synthesis of RNA probes by in vitro
   transcription by bacteriophage DNA-
   dependent RNA polymerase.
In vitro
transcriptio
n
Labeling the 5′ and 3′ termini of DNA
   Labeling the 3′ termini of double-stranded DNA
    using the Klenow fragment of E. coli DNA
    polymerase I. (lack of 5’  3’ exonuclease
    activity)
   Labeling the 3′ termini of double-stranded DNA
    using bacteriophage T4 DNA polymerase.
   Labeling the 5′ termini of DNA with
    bacteriophage T4 polynucleotide kinase.
T4 polynucleotide kinase
activity
Non-isotope labeling
 Digoxigenin-11-dUTP (DIG-dUTP) labeling
  - DNA labeling
  - Oligonucleotide labeling
  - RNA labeling
PCR Labeling, Random Primed
Labeling, and RNA Labeling
Prehybridization

 Add prehybridization solution and
  prehybridize at hybridization temperature for
  2-4 hr
Hybridization

            Remove prehybridization
             solution and add
             hybridization solution
            Add 500,000 cpm of the
             probe/ml hybridization
             solution.
            Hybridize overnight at
             appropriate temperature.
Post-hybridization washing

 Wash twice, 15 min each, in 1x SSC, 0.1% SDS at
  room temperature.
 Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS
  at hybridization temp
Critical parameters (II)
  Homology between the probe and the sequences
   being detected
    Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (%
     formamide)- 600/n - 1.5 (% mismatch)
    Factors can be changed:
      Hybridization temp.
      Washing temp.
      Salt concentration during washing
     High temp., low salt: high stringency
     Low temp., high salt: low stringency
    If 50 % formamide is used
      42 oC for 95 ~ 100 % homology
      37 oC for 90 ~ 95 % homology
      32 oC for 85 ~ 90 % homology
Comparison of nitrocellulose
and nylon membranes

    NC                   Nylon
    Hydrophobic binding Covalent binding
    Fragile              Durable
    Probe length > 200 ~ < 200 ~ 300 bp is
    300 bp               O.K.
    Lower background     Higher background
    Cannot be exposed    Can be exposed to
    to basic solution    basic solution
    Not easily           Can be reprobed
    reprobed             several times
Signals detection
 Autoradioragraphy
 Non-isotope detection system
  - Chemiluminescent detection
  - Colorimetric detection
  - Multicolor detection
Autoradiography

              Exposure to x-ray film
Northern blotting or Northern
hybridization
  Technique for detecting specific RNAs
   separated by electrophoresis by hybridization
   to a labeled DNA probe.
The flow chart of Northern
hybridization samples and run RNA gel
        Prepare RNA

                  Northern transfer

                  Probe preparation
                                           Isotope
                   Prehybridization        Non-isotope

                    Hybridization

              Post-hybridization washing

                   Signal detection
Preparation of
agarose/formaldehyde gel
  E.g. Prepare a 350 ml 1.2%
   agarose/formaldehyde gel
    4.2 g agarose in 304.5 g water. Microwave, then
     cool to 60°C. Add 35 ml 10x MOPS running buffer
     and 10.5 ml 37% formaldehyde
Preparation of RNA samples
 Prepare a premix:
    5 µl of 10x MOPS running buffer
    8.75 µl of 37% formaldehyde
    25 µl of formamide.
 Prepare RNA samples:
    38.75 µl of premix
    RNA (0.5 to 10 µg)*
    water to 50 µl
 *If the mRNA species of interest makes up a relatively high percentage of
  the mRNA in the cell (>0.05% of the message), total cellular RNA can be
  used. If the mRNA species of interest is relatively rare, however, it is
  advisable to use poly(A)+ RNA.
 Incubate 15 min at 55°C
Running the RNA gel

 Add 10 µl formaldehyde loading buffer to
  each sample and load gel. Run gel at 100 to
  120 V for ~3hr.
 Remove gel from the running tank and rinse
  several times in water. Place gel in 10x SSC
  for 45 min.
 Do not need post-transferring gel treatment
An example of Northern
 blotting
Northern blot



  RNA gel                 28 S
                          18 S
Western blotting, or
immunoblotting

Technique for detecting specific proteins
separated by electrophoresis by use of
labeled antibodies.
Flow chart of Western blotting
                   Electrophoresing the protein sample

                 Assembling the Western blot sandwich

          Transferring proteins from gel to nitrocellulose paper

                     Staining of transferred proteins

      Blocking nonspecific antibody sites on the nitrocellulose paper

          Probing electroblotted proteins with primary antibody

         Washing away nonspecifically bound primary antibody

Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and
             formation of a diaminobenzidine (DAB) precipitate

                     Photographing the immunoblot
SDS polyacrylamide-gel
electrophoresis (SDS-PAGE)
Analysis of protein samples by SDS
polyacrylamide-gel electrophoresis and
Western blotting


                                      Protein bands
                                      detected by
                                      specific antibody




       SDS-PAGE        Western blot
Comparison of Southern, Northern,
and Western blotting techniques
                  Southern blotting   Northern blotting     Western blotting
   Molecule          DNA (ds)           mRNA (ss)              Protein
   detected
      Gel           Agarose gel         Formaldehyde       Polyacrylamide gel
electrophoresis                          agarose gel
      Gel          Depurination,              -                     -
 pretreatment   denaturation, and
                   neutralization
Blotting method Capillary transfer    Capillary transfer    Electric transfer
     Probes            DNA               cDNA, cRNA         primary antibody
                  Radioactive or        Radioactive or
                  nonradioactive        nonradioactive
   Detection     Autoradiography       Autoradiography     Chemiluminescent
     system     Chemiluminescent      Chemiluminescent       Colorimetric
                    Colorimetric         Colorimetric

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Southern northern and western blotting

  • 2. Comparison of Southern, Northern, and Western analyses of Gene X
  • 3. Southern hybridization  First described by E. M. Southern in 1975.  Applications of Southern hybridization  RFLP’s, VNTR’s and DNA fingerprinting  Checking of the gene knockout mice  The flow chart of Southern hybridization
  • 4. Southern hybridization Transfer buffer
  • 5. Detection of an RFLP by Southern blotting
  • 6. Detection of the sickle-cell globin gene by Southern blotting
  • 7. Checking of the gene knockout mice
  • 8. Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Isotope Non-isotope Prehybridization Hybridization Post-hybridization washing Signal detection
  • 9. Preparing the samples and running the gel  Digest 10 pg to 10 µg of desired DNA samples to completion.  Prepare an agarose gel, load samples (remember marker), and electrophorese.  Stain gel ethidium bromide solution (0.5 µg/ml).  Photograph gel (with ruler).
  • 10. Critical parameters (I)  Note the complexity of DNA  Genomic DNA  A single-copy of mammalian gene, 3 Kb average in length 10 µg x 3 Kb/3 x 106 Kb = 10 µg x 1/106 = 10 pg  Plasmid DNA or PCR products 0.1 µg of a 3 Kb plasmid DNA ≅100 ng
  • 11. Gel treatment  Acid treatment  0.2 N HCl solution  Denaturation  NaOH solution  Neutralization  Tris-Cl buffer (pH8.0)
  • 12. Southern transfer  Measure gel and set up transfer assembly:  Wick in tray with 20x SSC  Gel  Nitrocellulose or Nylon filters (soaked in H2O and 20x SSC)  3MM Whatman filter paper  Paper towels  Weight
  • 13. After Southern transfer  Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC  Bake nitrocellulose at 80°C for 2 hr or UV-crosslink Nylon membrane for seconds
  • 14. Preparation of probes  Synthesis of uniformly labeled double-stranded DNA probes  Preparation of single-stranded probes  Labeling the 5′ and 3′ termini of DNA
  • 15. Synthesis of double-stranded DNA probes - Nick translation of DNA - Labeled DNA probes using random oligonucleotide primers
  • 17. Preparation of single-stranded probes  Synthesis of single-stranded DNA probes using bacteriophage M13 vectors.  Synthesis of RNA probes by in vitro transcription by bacteriophage DNA- dependent RNA polymerase.
  • 19. Labeling the 5′ and 3′ termini of DNA  Labeling the 3′ termini of double-stranded DNA using the Klenow fragment of E. coli DNA polymerase I. (lack of 5’  3’ exonuclease activity)  Labeling the 3′ termini of double-stranded DNA using bacteriophage T4 DNA polymerase.  Labeling the 5′ termini of DNA with bacteriophage T4 polynucleotide kinase.
  • 21. Non-isotope labeling  Digoxigenin-11-dUTP (DIG-dUTP) labeling - DNA labeling - Oligonucleotide labeling - RNA labeling
  • 22. PCR Labeling, Random Primed Labeling, and RNA Labeling
  • 23. Prehybridization  Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr
  • 24. Hybridization  Remove prehybridization solution and add hybridization solution  Add 500,000 cpm of the probe/ml hybridization solution.  Hybridize overnight at appropriate temperature.
  • 25. Post-hybridization washing  Wash twice, 15 min each, in 1x SSC, 0.1% SDS at room temperature.  Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS at hybridization temp
  • 26. Critical parameters (II)  Homology between the probe and the sequences being detected  Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (% formamide)- 600/n - 1.5 (% mismatch)  Factors can be changed:  Hybridization temp.  Washing temp.  Salt concentration during washing High temp., low salt: high stringency Low temp., high salt: low stringency  If 50 % formamide is used  42 oC for 95 ~ 100 % homology  37 oC for 90 ~ 95 % homology  32 oC for 85 ~ 90 % homology
  • 27. Comparison of nitrocellulose and nylon membranes NC Nylon Hydrophobic binding Covalent binding Fragile Durable Probe length > 200 ~ < 200 ~ 300 bp is 300 bp O.K. Lower background Higher background Cannot be exposed Can be exposed to to basic solution basic solution Not easily Can be reprobed reprobed several times
  • 28. Signals detection  Autoradioragraphy  Non-isotope detection system - Chemiluminescent detection - Colorimetric detection - Multicolor detection
  • 29. Autoradiography  Exposure to x-ray film
  • 30. Northern blotting or Northern hybridization  Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.
  • 31. The flow chart of Northern hybridization samples and run RNA gel Prepare RNA Northern transfer Probe preparation Isotope Prehybridization Non-isotope Hybridization Post-hybridization washing Signal detection
  • 32. Preparation of agarose/formaldehyde gel  E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel  4.2 g agarose in 304.5 g water. Microwave, then cool to 60°C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde
  • 33. Preparation of RNA samples  Prepare a premix:  5 µl of 10x MOPS running buffer  8.75 µl of 37% formaldehyde  25 µl of formamide.  Prepare RNA samples:  38.75 µl of premix  RNA (0.5 to 10 µg)*  water to 50 µl  *If the mRNA species of interest makes up a relatively high percentage of the mRNA in the cell (>0.05% of the message), total cellular RNA can be used. If the mRNA species of interest is relatively rare, however, it is advisable to use poly(A)+ RNA.  Incubate 15 min at 55°C
  • 34. Running the RNA gel  Add 10 µl formaldehyde loading buffer to each sample and load gel. Run gel at 100 to 120 V for ~3hr.  Remove gel from the running tank and rinse several times in water. Place gel in 10x SSC for 45 min.  Do not need post-transferring gel treatment
  • 35. An example of Northern blotting Northern blot RNA gel 28 S 18 S
  • 36. Western blotting, or immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.
  • 37. Flow chart of Western blotting Electrophoresing the protein sample Assembling the Western blot sandwich Transferring proteins from gel to nitrocellulose paper Staining of transferred proteins Blocking nonspecific antibody sites on the nitrocellulose paper Probing electroblotted proteins with primary antibody Washing away nonspecifically bound primary antibody Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate Photographing the immunoblot
  • 39. Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blotting Protein bands detected by specific antibody SDS-PAGE Western blot
  • 40. Comparison of Southern, Northern, and Western blotting techniques Southern blotting Northern blotting Western blotting Molecule DNA (ds) mRNA (ss) Protein detected Gel Agarose gel Formaldehyde Polyacrylamide gel electrophoresis agarose gel Gel Depurination, - - pretreatment denaturation, and neutralization Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA cDNA, cRNA primary antibody Radioactive or Radioactive or nonradioactive nonradioactive Detection Autoradiography Autoradiography Chemiluminescent system Chemiluminescent Chemiluminescent Colorimetric Colorimetric Colorimetric