Blotting methods

1. Blotting

2. What is Blotting?
Transfer of protein, rna or dna molecules from a relatively
thick acrylamide or agarose gel or to a paper like
membrane (usually nylon or nitrocellulose) by capilliarity
or an electric field, preserving the spatial arrangement.
Different blotting is used to detect different type of
macromolecules such as southern blotting is used for DNA
analysis, western blotting is for protein analysis, northern
blotting is for RNA analysis and eastern for carbohydrate
detection.

3. Southern, Northern and
Western blotting

4. Comparison of Southern, Northern, and
Western analyses of Gene X

5. DNA
• Each individuals unique genetic blueprint is
stored in material known as DNA.
• DNA is found in all cells containing a nucleus.
• DNA can be extracted for analysis from hair,
bones, saliva, sperm, skin, organs, all body
tissues and blood.

6. DNA
• The deoxyribonucleicacid, DNA, is a long
chain of nucleotides which consist of:
• 1. Deoxyribose(sugar with 5 carbons)
• 2. Phosphate groups
• 3. Organic(nitrogenous)bases

7. DNA STORAGE AND COLLECTION
• I. Temperature Storage for DNA
–Purified DNA may be refrigerated at
4°C for up to 3 years.
–Samples kept over 3 years should be
frozen at -70°C.

8. DNA STORAGE AND COLLECTION
• II. Specimens used in DNA testing
–Whole blood
–Solid tissue
–Serum and plasma
–Urine
–Bone marrow
–and many others

9. DNA STORAGE AND COLLECTION
• III. Specimen Collection Requirements
–A. Blood and Bone Marrow
• Collection tubes are EDTA or ACD
• 5-15 ml
• Samples should not be frozen for transport
• 4-25°C

10. DNA STORAGE AND COLLECTION
• B. Serum
–Collection tubes with no additives
–100 µl to 1 ml
–Transported at 20-25°C

11. DNA STORAGE AND COLLECTION
• Spin the samples to separate the plasma, RBC,
and buffy coat.
• Extract the buffy coat
• The buffy coat is used because the WBC are
nucleated and contain DNA.

12. DNA STORAGE AND COLLECTION
• C. Tissue
–A sterile container with no formalin or
paraffin must be used for collection.
–30 mg
–Dry ice should be used for transport.

13. DNA STORAGE AND COLLECTION
• D. Urine
–Urine container should be used for
collection.
–At least 1 ml should be collected.
–Transported at 4-25°C

14. SOUTHERN BLOTTING
• The technique was developed by E.M.
Southern in 1975.
• The Southern blot is used to detect the
presence of a particular piece of DNA in a
sample.
• The DNA detected can be a single gene, or it
can be part of a larger piece of DNA such as a
viral genome.

15. Southern hybridization
Transfer buffer

16. Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope

17. Preparing the samples and running the
gel
• Digest 10 pg to 10 g of desired DNA samples
to completion.
• Prepare an agarose gel, load samples
(remember marker), and electrophorese.
• Stain gel ethidium bromide solution (0.5
g/ml).
• Photograph gel (with ruler).

18. Critical parameters (I)
• Note the complexity of DNA
–Genomic DNA
• A single-copy of mammalian gene, 3 Kb
average in length
10 g x 3 Kb/3 x 106 Kb = 10 g x 1/106 =
10 pg
–Plasmid DNA or PCR products
0.1 g of a 3 Kb plasmid DNA 100 ng

19. Gel treatment
• Acid treatment
– 0.2 N HCl solution
• Denaturation
– NaOH solution
• Neutralization
– Tris-Cl buffer (pH8.0)

20. 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

21. 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

22. Preparation of probes
• Synthesis of uniformly labeled double-
stranded DNA probes
• Preparation of single-stranded probes
• Labeling the 5 and 3 termini of DNA

23. Synthesis of double-stranded DNA probes
- Nick translation of DNA
- Labeled DNA probes using random
oligonucleotide primers

24. Nick translation

25. 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.

26. In vitro
transcription

27. • Labeling the 3 termini of double-stranded
DNA using the Klenow fragment of E. coli
DNA polymerase I. (lack of 5’  3’
exonucleaseactivity)
• Labeling the 3 termini of double-stranded
DNA using bacteriophage T4 DNA polymerase.
• Labeling the 5 termini of DNA with
bacteriophageT4 polynucleotide kinase.
Labeling the 5 and 3 termini of DNA

28. T4 polynucleotide kinase activity

29. Non-isotope labeling
• Digoxigenin-11-dUTP (DIG-dUTP) labeling
- DNA labeling
- Oligonucleotide labeling
- RNA labeling

30. PCR Labeling, Random Primed Labeling,
and RNA Labeling

31. Prehybridization
• Add prehybridization solution and
prehybridize at hybridization temperature for
2-4 hr

32. Hybridization
• Remove prehybridization
solution and add
hybridization solution
• Add 500,000 cpm of the
probe/ml hybridization
solution.
• Hybridize overnight at
appropriate temperature.

33. 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

34. 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

35. Comparison of nitrocellulose and nylon
membranes
NC Nylon
Hydrophobic binding Covalent binding
Fragile Durable
Probe length > 200 ~
300 bp
< 200 ~ 300 bp is
O.K.
Lower background Higher background
Cannot be exposed
to basic solution
Can be exposed to
basic solution
Not easily
reprobed
Can be reprobed
several times

36. Signals detection
• Autoradioragraphy
• Non-isotope detection system
- Chemiluminescent detection
- Colorimetric detection
- Multicolor detection

37. Autoradiography
• Exposure to x-ray
film

38. SOUTHERN BLOTTING
• The key to this method is hybridization.
• Hybridization-processof forming a double-
stranded DNA molecule between a single-
stranded DNA probe and a single-stranded
target patient DNA.

39. SOUTHERN BLOTTING
• There are 2 important features of
hybridization:
– The reactions are specific-the probes will only
bind to targets with a complementary sequence.
– The probe can find one molecule of target in a
mixture of millions of related but non-
complementary molecules.

41. SOUTHERN BLOTTING
• Steps for hybridization
– 1. The mixture of molecules is separated.
– 2. The molecules are immobilized on a matrix.
– 3. The probe is added to the matrix to bind to the
molecules.
– 4. Any unbound probes are then removed.
– 5. The place where the probe is connectedcorresponds to
the location of the immobilized target molecule.

42. SOUTHERN BLOTTING
• I. DNA Purification
–Isolate the DNA in question from the rest of
the cellular material in the nucleus.
–Incubate specimen with detergent to
promote cell lysis.
–Lysis frees cellular proteins and DNA.

43. SOUTHERN BLOTTING
• Proteins are enzymatically degraded by
incubation with proteinase.
• Organic or non-inorganic extraction removes
proteins.
• DNA is purified from solution by alcohol
precipitation.
• Visible DNA fibers are removed and
suspended in buffer.

44. SOUTHERN BLOTTING
• II. DNA Fragmentation
–Cut the DNA into different sized pieces.
–Use restriction endonucleases (RE)
–Bacterial proteins
–In vivo, they are involved in DNA
metabolism and repair or in bacterial host
defense.

45. SOUTHERN BLOTTING
• Nucleases hydrolyze the bonds that
connect bases within the strand,
resulting in cleavage of the strand.
• They cleave the double stranded nucleic
acid only at specific points.

46. SOUTHERN BLOTTING
• This allows for specific sequences to
be identified more readily.
• Fragments are now easily separated by
gel electrophoresis.

47. SOUTHERN BLOTTING
• III. Gel Electrophoresis
–Sorts the DNA pieces by size
–Gels are solid with microscopic pores
–Agarose or polyacrimide
–Gel is soaked in a buffer which controls the
size of the pores
–Standards should also be run

48. SOUTHERN BLOTTING
• Nucleic acids have a net negative charge and will move
from the left to the right. The larger molecules are held up
while the smaller ones move faster. This results in a
separation by size.

49. SOUTHERN BLOTTING
• Gels can be stained with ethidium bromide.
• This causes DNA to fluoresce under UV light
which permits photography of the gel.
• You can tell the exact migration of DNA
standards and the quality of the RE digestion
of the test DNA.

50. SOUTHERN BLOTTING
• High quality intact DNA should give the
appearance of a single band.
• Degraded material will smear downwards.
• Only a small amount of degradation is
tolerable.

51. SOUTHERN BLOTTING
• IV. Blotting
–Transfer the DNA from the gel to a solid
support.
–The blot is usually done on a sheet of
nitrocellulose paper or nylon.

52. SOUTHERN BLOTTING
• DNA is partially depurinated with dilute HCL
which promotes higher efficiency transfer by
breaking down fragments into smaller pieces.
• DNA is then denatured with an alkaline
solution such as NAOH.
• This causes the double stranded to become
single-stranded.

53. SOUTHERN BLOTTING
• DNA is then neutralized with NaCl to
prevent re-hybridization before adding the
probe.
• Transferred by either electrophoresis or
capillary blotting.

54. SOUTHERN BLOTTING
• 1) Electrophoresis- takes advantage of the
molecules negative charge.

55. SOUTHERN BLOTTING
• 2) Capillary blotting-fragments are eluted from the gel and
deposited onto the membrane by buffer that is drawn
through the gel by capillary action.

56. SOUTHERN BLOTTING
• The blot is made permanent by:
–Drying at ~80°C
–Exposing to UV irradiation

57. SOUTHERN BLOTTING
• V
. Blocking
–Buffer binds to areas on the blot not
occupied by patient DNA.
–Blocks the empty sites from being bound
during hybridization.

58. SOUTHERN BLOTTING
• VI. Preparing the probe
–Small piece of DNA used to find another
piece of DNA
–Must be labeled to be visualized
–Usually prepared by making a radioactive
copy of a DNA fragment.

59. SOUTHERN BLOTTING
• The DNA fragment is labeled by the Random
Hexamer Labeling Process:
– 1. The template DNA is denatured by boiling.
– 2. A mixture of hexamers (6 nucleotides)
containing all possible sequences is added and
allow to base pair.
– 3. DNA polymerase is added with radioactive
nucleotides.
– 4. The mixture is boiled to separate the strands
and is ready for hybridization.

60. SOUTHERN BLOTTING
• The Random Hexamer Labeling Process
produces a radioactive single-stranded
DNA copy of both strands of the
template for use as a probe.

61. SOUTHERN BLOTTING

62. SOUTHERN BLOTTING
• VII. Hybridization
–The labeled probe is added to the blocked
membrane in buffer and incubated for
several hours to allow the probe molecules
to find their targets.

63. SOUTHERN BLOTTING
• VIII. Washing
– Excess probe will have bound nonspecifically to
the membrane despite the blocking reagents.
– Blot is incubated with wash buffers containing
NaCl and detergent to wash away excess probe
and reduce background.

64. SOUTHERN BLOTTING
• IX. Detection
–Radioactive probes enable autoradiographic
detection.

65. SOUTHERN BLOTTING
• If the probe is radioactive, the particles it
emits will expose X-ray film.
• By pressing the filter and film, the film will
become exposed wherever probe is bound to
the filter.
• After development, there will be dark spots on
the film wherever the probe bound.

66. USES
• Identify mutations, deletions, and gene
rearrangements
• Used in prognosis of cancer and in prenatal
diagnosis of genetic diseases
• Leukemias
• Diagnosis of HIV-1 and infectious disease

67. USES
• Every person has repeated sequences of base
pairs which are called Variable Number
Tandem Repeats (VNTRs)
• To find a particular VNTR we use a radioactive
version of the one in question.
• This pattern is known as a DNA fingerprint.

68. USES
• Applications of DNA fingerprinting include:
– Paternity and Maternity Testing
– Criminal Identification and Forensics
– Personal Identification

69. Northern blotting or Northern
hybridization
• Technique for detecting specific RNAs
separated by electrophoresis by hybridization
to a labeled DNA probe.

70. The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope

71. 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

72. Preparation of RNA samples
• Prepare a premix:
– 5 l of 10x MOPS running buffer
– 8.75 l of 37% formaldehyde
– 25 l of formamide.
• Prepare RNA samples:
– 38.75 l of premix
– RNA (0.5 to 10 g)*
– water to 50 l
• *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.
• Incubate15 min at 55C

73. Running the RNA gel
• Add 10 l 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

74. An example of Northern blotting
Northern blot
RNA gel 28 S
18 S

75. Western blotting, or immunoblotting
T
echnique for detecting specific proteins
separated by electrophoresis by use of
labeled antibodies.

76. Flow chart of Western blotting
Electrophoresingthe protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulosepaper
Stainingof transferred proteins
Blocking nonspecific antibodysites on the nitrocellulose paper
Probing electroblotted proteinswith primary antibody
Washing away nonspecifically bound primaryantibody
Detecting bound antibodyby horseradish peroxidase-anti-Igconjugate and
formation of a diaminobenzidine (DAB) precipitate
Photographingthe immunoblot

77. SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

78. Analysis of protein samples by SDS polyacrylamide-gel
electrophoresis and Western blotting
Protein bands
detected by
specific antibody
SDS-PAGE Western blot

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