Applications of antibody in

1
Applications of antibody in biomedical
research/proteomics
Jau-Song Yu (???) Department of Cell and
Molecular Biology Medical College of Chang Gung
University
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Cell cycle analysis of cultured newt lung cells
3
Structure of antibody
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Classes of antibody
5
How specific antibody can be made ?
Sources of antigen/immunogen ---
GST-fusion protein
Native protein
Cell
Carrier hapten
6
What is an epitope ?
Antibodies are raised by the immune system
against regions on the surface of antigens known
as epitopes. An epitope, or antigenic
determinants, is defined as a region of amino
acid or carbohydrate residues to which an
antibody can bind. Continuous vs. discontinuous
epitopes
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Polyclonal vs. Monoclonal antibodies
Monoclonal Ab (mAb) ---- antibody
produced from a single clone of B cells
in immunized animal (mouse) (against only
one specific epitope) Polyclonal Ab ----
antibodies produced from multiple clones of B
cells in immunized animal (mouse,
rabbit, goat, horse) (against many
epitopes)
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Polyclonal vs. Monoclonal antibodies
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Anti-peptide antibodies
Carrier peptides (haptens)
(Polyclonal or Monoclonal antibodies ?)
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Generation of anti-peptide antibodies

Synthetic peptide
Cross-linked by glutaraldehyde
Anti-sera from immunized rabbits
Affinity-purified by resins coupled with peptide
H
L
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What applications of antibody ?
1. Western Blot --- detection of specific antigen
in tissue/cell extracts
Cell
Extracts
SDS-PAGE
E
Membrane
Blotting
Detection
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WB in A431 cell extracts
Anti-peptide Ab of human GSK3a (53 kDa)
462-475
Pre Ab
(- ) peptide
13
2. Immunoprecipitation--- quickly isolation of
specific antigen
from tissue/cell extracts
Cell
Extracts
cfg
b-ME/heating SDS-PAGE
H
L
Protein A/G-Beads
ppt sup
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Anti-peptide Ab of human PAK2 (62 kDa)
IP/WB using A431 cell extracts
ppt sup
ppt
- - peptide -
1-17
B
E
E
Biotin-strepavidin-AP
2nd Antibody-AP
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3. Immunofluorescence--- localization of specific
antigen in tissue/cells
detergent
fixation
Cell
1st Ab
2nd Ab
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Mouse mAb of human HSP 70 (70 kDa)
Control
Heat shock
HeLa cells
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Anti-peptide Ab of active caspase-3
Newborn rat brain
tissue/transient ischemia Double staining with PI
(red) and anti-active caspase-3 (green)
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4. Immunohistochemistry--- localization of
specific antigen in tissues
Anti-peptide Abs of FGF receptor 1-4
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In proteomic study
5. Identification of specific protein-protein
interaction by IP
C
C
B
B
E
E
D
D
cfg
ppt
b-ME/heating SDS-PAGE
B
IgG heavy chain
C
D
IgG light chain
E
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M IP Sup
To identify proteins that interact with a
specific gene product X in cells
200
116
97
A431 cell extracts (10 mg proteins) 5 mg of mAb
against X (32 kDa) incubation at 4oC for 1.5 h
incubation with protein A-beads at 4oC for 1.5
h cfg, wash, collect IP 12.5 SDS-PAGE,
Comassie blue stain MALDI-TOF MS analysis of
bands of interest
66
H
55
36
X
31
L
21
21
Identification of proteins in small quantity by
mass spectrometers
B
IgG heavy chain
C
D
IgG light chain
E
(?)
digested by trypsin
MALDI-TOF MS or LC/MSn analysis
Database search/mapping
Protein identified
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MALDI-TOF MS (Matrix-assisted
laser desorption/ionization-Time of flight)
(??????????-???????)
Solid probe
Time of Flight
m/z time of flight
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MS data from MALDI-TOF MS
(a)
(b)
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Protein identification by database search
using peptide mapping software
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?
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MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG
LIKQIGENLI VPGGVKTIEA HSRMVIPGGI DVHTRFQMPD
QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF
DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG
VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRD IGAIAQVHAE
NGDIIAEEQQ RILDLGITGP EGHVLSRPEE VEAEAVNRAI
TIANQTNCPL YITKVMSKSS AEVIAQARKK GTVVYGEPIT
ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL
LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTL IPEGTNGTEE
RMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR
IAVGSDADLV IWDPDSVKTI SAKTHNSSLE YNIFEGMECR
GSPLVVISQG KIVLEDGTLH VTEGSGRYIP RKPFPDFVYK
RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK
TSPAKQQAPP VRNLHQSGFS LSGAQIDDNI PRRTTQRIVA
PPGGRANITS LG
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Direct sequencing of peptides from target
proteins by MALDI-TOF/TOF MS
spectrometry
MW. 2169
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Peptide fragmentation in MS/MS analysis of
specific peptide
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Direct sequencing of peptides from target
proteins by LC/MS/MS
spectrometry
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6. Identification of pathway-involving proteins
by specific Abs
P-Tyr Ab P-Thr Ab etc.
----Tyr----------
Phospho-specific antibodies ------
PO4
(Cell Signaling Tech.)
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P-Tyr Ab P-Thr Ab etc.
----Thr----------
Phospho-specific antibodies ------
PO4
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Nitro-Tyrosine specific antibodies --- NO pathway
----Tyr----------
NO
3T3 C6 A431 HeLa 3T3
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Acetylated-Lysine specific antibodies ---
Acetylation pathway
----Lys--------
C2H5
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Phospho-(Thr) MAPK/CDK Substrate Antibody ---
MAPK/CDK pathway
----Thr-Pro-------
PO4