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