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
transcription
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
Prepare RNA samples and run RNA gel

Northern transfer

Isotope
Probe preparation
Non-isotope

Prehybridization

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