Файл: Cell and Molecular Biology [High-Yield].pdf

118

CHAPTER 14

Polyacrylamide gel

Nitrocellulose

membrane

Film

Hear

t RNA

Br

ain RNA

Placenta RNA

Lung RNA

Transfer

Hybridization

Autoradiography

P

32

-radiolabeled

cDNA probe

Northern blot

● Figure 14-10

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119

MOLECULAR BIOLOGY TECHNIQUES

Northern Blot (mRNA):

• A Northern blot is a variant of a Southern blot that separates undigested mRNA based on size by

PAGE.

• A Northern blot is used to determine whether a gene is being expressed or not within a certain

tissue by analyzing mRNA levels.

• The variable-sized mRNAs are transferred to nitrocellulose membrane and then hybridized with

a radiolabeled P

32

cDNA probe. The P

32

cDNA probe is identical to the template DNA strand

that produced the mRNA.

• Photographic film is then placed over the nitrocellulose membrane, exposed, and developed

(autoradiography).

• Radiolabeled cDNA probe hybridization to specific mRNAs is documented by dark bands on the

photographic film.

• A real Northern blot using a radiolabeled cDNA probe for the FMR1 gene (Fragile X mental

retardation syndrome). Note that the highest levels of mRNA coded by the FMR1 gene are found
in the brain and testes (4.4 kb). Smaller transcripts of the mRNA (1.4 kb) are found in the heart.
This means that the FMR1 gene is expressed most highly in the brain and testes.

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CHAPTER 14

Polyacrylamide gel

Nitrocellulose

membrane

Protein cell line #1

Protein cell line #2

Cell line #1

Cell line #2

Protein cell line #3

Protein cell line #4

Transfer

Incubation

HRP-labeled secondary antibody
Chromogenic or
Chemiluminescent substrate

Specific
antibody

Western blot

● Figure 14-11

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121

MOLECULAR BIOLOGY TECHNIQUES

Western Blot (Protein):

• A Western blot separates proteins from various cell lines (1–4) based on size by PAGE.
• The proteins are transferred to a nitrocellulose membrane and then incubated with an antibody

to a specific protein.

• Antibody binding to specific proteins may be detected by a horseradish peroxidase

(HRP)–labeled secondary antibody which can be reacted with a chromogenic substrate (usual-
ly brown in color).

• A recent improvement in the technique uses a chemiluminescent substrate. In this case, anti-

body binding to specific proteins may be detected by an HRP–labeled secondary antibody which
can be reacted with a chemiluminescent substrate. The chemiluminescent substrate allows
detection sensitivities superior to that of a chromogenic substrate, whereby picogram protein
levels of detection can be achieved using either X-ray film or imaging equipment.

• Antibody binding to specific proteins is documented by colored bands on the nitrocellulose

membrane (using a chromogenic substrate shown) or X-ray film (using a chemiluminescent sub-
strate not shown).

• A real Western blot shows a composite of six different Western blots for two different cell lines

(1 and 2). Antibody binding to specific proteins (i.e., claudin-1, claudin-2, claudin-4, claudin-7,
occludin, and ZO-1) is detected by an HRP and a chromogenic substrate. Note that the expres-
sion of the proteins is similar in both cell line 1 and cell line 2, except that claudin-2 appears to
be more highly expressed in cell line 1 than in cell line 2.

• Positively charged histone proteins. In general, most proteins are negatively charged and there-

fore can be separated using “normal” SDS-PAGE conditions which produce anionic SDS-protein
complexes. However, histones are positively charged and therefore Acid-Urea-PAGE conditions
are used to run positively charged proteins such as histones. Urea keeps the histones from form-
ing aggregates. In addition, the electric field needs to be reversed so that the positively charged
histones will migrate toward the bottom of the gel.

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122

CHAPTER 14

HRP

HRP

HRP

HRP

gp120

p24

p65

p51

Patient

#1 serum

Nitrocellulose

with separated

HIV proteins

HRP

Patient

#2 serum

HRP

Patient

#3 serum

HRP

Patient

#4 serum

HRP

gp160

  • gp120

  • gp41

p55
(gag precursor)

  • p24

  • p17

  • p9
  • p7

p65
(Protease)

p66
p51
(Reverse
transcriptase)

p31
(Integrase)

RNA

Lipid

envelope

eav gene

A

HRP-labeled
secondary
antibody

Cross-reactive
Abs

HIV Proteins

COLORMETRIC

REACTION

SUBSTRATE

PLATE

SERUM

3

2

1

B

gp160

p65
p55

p24

p17

gp160

p63

p51

HP

gp160

gp120

gp41

p51

p55

p65

p31

p24

p17

LP

I

N

C

gag gene

pol gene

● Figure 14-12

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