Applications of antibody in biomedical research/proteomics
Jau-Song Yu (余兆松 )
Department of Cell and Molecular BiologyMedical College of Chang Gung University
Cell cycle analysis of cultured newt lung cells
Structure of antibody
Classes of antibody
How specific antibody can be made ?
Sources of antigen/immunogen ---
GST-fusion protein
Carrier + hapten
Native proteinCell
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 antibodycan bind.
Continuous vs. discontinuous epitopes:
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)
Polyclonal vs. Monoclonal antibodies
Anti-peptide antibodies
Carrier + peptides (haptens)
(Polyclonal or Monoclonal antibodies ?)
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
What applications of antibody ?
1. Western Blot --- detection of specific antigen in tissue/cell extracts
Cell Extracts SDS-PAGE
Membrane
E
BlottingDetection
Anti-peptide Ab of human GSK3(53 kDa)
462-475
Pre Ab(- +) peptide
WB in A431 cell extracts
2. Immunoprecipitation--- quickly isolation of specific antigen from tissue/cell extracts
Cell Extracts
Protein A/G-Beads
cfg-ME/heatingSDS-PAGE
ppt sup
H
L
Anti-peptide Ab of human PAK2(62 kDa)
1-17
IP/WB using A431 cell extracts
ppt sup- + - + peptide + -
E
B
E
ppt
Biotin-strepavidin-AP2nd Antibody-AP
3. Immunofluorescence--- localization of specific antigen in tissue/cells
Cell
fixation detergent
1st Ab
2nd Ab
Mouse mAb of human HSP 70(70 kDa)
Heatshock
Control
HeLa cells
Anti-peptide Ab of active caspase-3
Newborn rat brain tissue/transient ischemiaDouble staining with PI (red) and anti-active caspase-3 (green)
4. Immunohistochemistry--- localization of specific antigen in tissues
Anti-peptide Abs of FGF receptor 1-4
BC
D
BeadProtein A/G BeadProtein A/G
B
-ME/heatingSDS-PAGE
IgG heavy chain
B
C
D
CD
IgG light chain
5. Identification of specific protein-protein interaction by IP
E E
E
In proteomic study
cfg
ppt
M IP Sup To identify proteins that interactwith a specific gene product X in cells
A431 cell extracts (10 mg proteins)
5 g 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
H
L
X36
31
55
66
21
97116
200
MALDI-TOF MS or LC/MSn analysis
digested by trypsin
Database search/mapping
(?)
Protein identified
IgG heavy chain
B
CD
IgG light chainE
Identification of proteins in small quantity by mass spectrometers
First DetectorLaser
Reflector
Second Detector
圖十一:在 MALDI-TOF MS中的反射器設計
MALDI-TOF MS(Matrix-assisted laser desorption/ionization-Time of flight) (基質輔助雷射脫附游離 -飛行時間質譜儀 )
Time of Flight
Solid probe
m/z ~ time of flight
(a)
(b)
MS data from MALDI-TOF MS
Protein identification by database search using peptide mapping software
?
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
Direct sequencing of peptides from target proteins by MALDI-TOF/TOF MS spectrometry
MW. 2169
Peptide fragmentation in MS/MS analysis of specific peptide
Direct sequencing of peptides from target proteins by LC/MS/MS spectrometry
6. Identification of pathway-involving proteins by specific Abs
Phospho-specific antibodies ------P-Tyr AbP-Thr Abetc.
(Cell Signaling Tech.)
----Tyr----------
PO4
Phospho-specific antibodies ------P-Tyr AbP-Thr Abetc.
----Thr----------
PO4
Nitro-Tyrosine specific antibodies --- NO pathway
3T3 C6 A431 HeLa 3T3
----Tyr----------
NO
Acetylated-Lysine specific antibodies --- Acetylation pathway
----Lys--------
C2H5
Phospho-(Thr) MAPK/CDK Substrate Antibody --- MAPK/CDK pathway
----Thr-Pro-------
PO4