MCB 730: Southern Blot Lab
• Southern, Northern & Western blotting
• Background
• Overview
• Detailed Protocol
Who is this guy?
Sir Edwin Mellor Southern of course (1938- )
• Inventor of the Southern blot
• http://en.wikipedia.org/wiki/Edwin_Southern
Characterization: Southern blot
hybridization
-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-developed by Edwin Southern
Characterization: Northern blot
hybridization
X RNA
X
x
salt
X
RNA
-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-reveals mRNA size (and approximate protein size), tissue- and organspecific expression, and kinetic patterns of expression
Characterization: Western blotting
X Protein
Enzyme
reaction
or
X
React with
Antibody
X
x
Buffer; requires electric current
X
-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)
-requires the use of an electric current to facilitate transfer
DNA Microarrays:
An Introduction
Microarray
Result:
Much analysis
to follow
Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-developed by Edwin Southern
Southern blotting of genomic DNA
Steps
1. Extraction of genomic DNA
2. Electrophoresis of genomic DNA (or more commonly
restriction enzyme-digested genomic DNA)
3. Before blotting, treat the gel with 0.2N HCl, denaturation and
neutralization solution
4. Blotting- Transfer gel to the nitrocellulose membrane by
capillary action
A) Sponge method
B) Wick method
5. After the transfer, cross-link the DNA to the nitrocellulose
membrane
A. UV- cross linking
B. Bake at 120°C for 30 min
C. Bake at 80°C for 2 hrs
6. Making the probe
Label the probe to be hybridized using radioactive or
non-radioactive methods
Non-radioactive methods A) Colorimetric
B) Chemiluminescent
* Roche DIG- DNA labeling (Non-radioactive) kits are used
for detection
DIG or Digoxigenin is used for labeling the probe. DIG
steroid found exclusively in the flowers and leaves of the
plants Digitalis purpurea, Digitalis orientalis and Digitalis
lanata (foxgloves).
(see http://en.wikipedia.org/wiki/Digoxigenin)
DIG-11-dUTP replaces dTTP in the random primed DNA
labeling reaction.
DIG-DNA Labeling: DNA is random primed labeled with
Digoxigenin-11-dUTP using DIG-High Prime, a 5x
concentrated labeling mixture of random hexamers, dNTP
mix containing alkali-labile Digoxigenin-11-dUTP, labeling
grade Klenow enzyme and an optimized reaction buffer.
Structure of digoxigenin- and biotin-modified nucleotides
Note that the digoxigenin and biotin groups in these examples are linked to the 5′ carbon
atom of the uridine of dUTP by spacer groups consisting respectively of a total of 11 carbon
atoms (digoxigenin-11-UTP) or 16 carbon atoms (biotin-16-dUTP). The digoxigenin and
biotin groups are reporter groups: after incorporation into a nucleic acid they are bound by
specific ligands containing an attached marker such as a fluorophore.
Arabidopsis T-DNA mutant
LB
T-DNA
RB
Gene
1000bp probe
LB
RB
T-DNA
Disrupted gene
7. Prehybridization and hybridization of probe to the
membrane at the hybridization temperature.
8. Washes and immunological detection
Detection after stringency washes using anti-digoxigenin
antibody conjugated to AP (Alkaline phosphatase)
Colorimetric detection using NBT/ BCIP
BCIP is colorless and is dephosphorylated by AP
Dephosphorylated BCIP is oxidized by NBT
Oxidized BCIP will turn into a dark blue indigo dye
NBT is reduced to a dark blue dye
BCIP and NBT: additional information
•
•
•
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) is a chemical compound used in immunoblotting, in situ
hybridization, and immunohistochemistry, with nitro blue tetrazolium chloride (NBT), for sensitive
colorimetric detection of alkaline phosphatase. NBT serves as the oxidant (and gives also dark blue dye)
and BCIP is the alkaline phosphatase substrate. Alkaline phosphatase is commonly conjugated to
secondary antibodies.
BCIP (colorless)oxidation→ blue precipitate. BCIP-NBT naturally forms this bluish purple precipitate over
time; however, alkaline phosphatase speeds up the process 1000 fold. BCIP binds very tightly in the
alkaline phosphatase active site, but when NBT reacts with BCIP, it is released from the enzyme and the
colored precipitate forms.
Nitro blue tetrazolium is a chemical compound composed of two tetrazole moieties. It is used in
immunology for sensitive detection of alkaline phosphatase (with BCIP). NBT serves as the oxidant and
BCIP is the AP-substrate (and gives also dark blue dye). In immunohistochemistry the alkaline
phosphatase is often used as a marker, conjugated to an antibody. The colored product can either be of
the NBT/BCIP reaction reveals where the antibody is bound, or can be used in immunofluorescence.
Colorimetric blot
Chemiluminescent detection using CSPD
• Dephosphorylation of CSPD by alkaline phosphatase to
the metastable phenolate anion.
• Phenolate anion decomposes and emits light at a
wavelength of 477 nm.
• Light emission is captured on X-rays.
•
•
chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD), formally disodium 3-(4methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate
is a chemical substance with formula C18H20ClO7PNa2. It is a component of enhanced
chemiluminescence enzyme-linked immunosorbent assay (ELISA) kits, used for the detection of
minute amounts of various substances.
In typical uses of ELISA kits, the enzyme alkaline phosphatase removes the phosphate group
from CSPD, yielding a reactive anion (phenolate, which then splits itself in two components,
adamantane and 1,2-dioxetane. This second reaction emits turquoise-coloured light (λmax = 477
nm). The decomposition of the dioxetane generates a secondary glow.