Biotechnology and Genetic Engineering-PBIO 450/550

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Dec 14, 2012 (4 years and 6 months ago)

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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

-
transfer of
RNA

from a gel to a membrane (e.g., nitrocellulose, nylon)

-
reveals mRNA size (and approximate protein size), tissue
-

and organ
-

specific expression, and kinetic patterns of expression

X

RNA

X

x

salt

X

RNA

Characterization: Western blotting

-
transfer of
protein

from a gel to a membrane (e.g., nitrocellulose, nylon)

-
requires the use of an electric current to facilitate transfer

X

Protein

X

x


Buffer; requires electric current

X

React with

Antibody

X

Enzyme

reaction

or

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


T
-
DNA

Gene

RB

LB


T
-
DNA

Disrupted

gene

RB

LB

1000bp probe

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)









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




Colorimetric detection using NBT/ BCIP


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
.





BCIP and NBT: additional information

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
-
(4
-
methoxyspiro{1,2
-
dioxetane
-
3,2'
-
(5'
-
chloro)tricyclo[3.3.1.1
3,7
]decan}
-
4
-
yl)phenyl phosphate

is a
chemical substance

with formula C
18
H
20
ClO
7
PNa
2
. 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 (
λ
max

= 477
nm). The decomposition of the dioxetane generates a secondary glow.