1. Applications of antibody in biomedical research/proteomics Jau-Song Yu ( 余兆松 ) Department of Cell and Molecular Biology Medical College of Chang Gung University

2. Cell cycle analysis of cultured newt lung cells

3. Structure of antibody

4. Classes of antibody

5. How specific antibody can be made ? Sources of antigen/immunogen --- GST-fusion protein Carrier + hapten Native protein Cell

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:

7. 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)

8. Polyclonal vs. Monoclonal antibodies

9. Anti-peptide antibodies Carrier + peptides (haptens) (Polyclonal or Monoclonal antibodies ?)

10. 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

11. What applications of antibody ? 1. Western Blot --- detection of specific antigen in tissue/cell extracts CellExtractsSDS-PAGE Membrane E BlottingDetection

12. Anti-peptide Ab of human GSK3  (53 kDa) 462-475 Pre Ab (- +) peptide WB in A431 cell extracts

13. 2. Immunoprecipitation--- quickly isolation of specific antigen from tissue/cell extracts CellExtracts Protein A/G-Beads cfg  -ME/heating SDS-PAGE ppt sup H L

14. 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-AP2 nd Antibody-AP

15. 3. Immunofluorescence--- localization of specific antigen in tissue/cells Cell fixation detergent 1 st Ab 2 nd Ab

16. Mouse mAb of human HSP 70 (70 kDa) Heat shock Control HeLa cells

17. Anti-peptide Ab of active caspase-3 Newborn rat brain tissue/transient ischemia Double staining with PI (red) and anti-active caspase-3 (green)

18. 4. Immunohistochemistry--- localization of specific antigen in tissues Anti-peptide Abs of FGF receptor 1-4

19. B C D Bead Protein A/G Bead Protein A/G B  -ME/heating SDS-PAGE IgG heavy chain B C D C D IgG light chain 5. Identification of specific protein-protein interaction by IP EE E In proteomic study cfg ppt

20. M IP Sup To identify proteins that interact with a specific gene product X in cells A431 cell extracts (10 mg proteins) 5  g of mAb against X (32 kDa) incubation at 4 o C for 1.5 h incubation with protein A-beads at 4 o C 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 X 36 31 55 66 21 97 116 200

21. MALDI-TOF MS or LC/MS n analysis digested by trypsin Database search/mapping (?) Protein identified IgG heavy chain B C D IgG light chain E Identification of proteins in small quantity by mass spectrometers

22. 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

23. (a) (b) MS data from MALDI-TOF MS

24. Protein identification by database search using peptide mapping software

25. ?

26. 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

27. Direct sequencing of peptides from target proteins by MALDI-TOF/TOF MS spectrometry MW. 2169

28. Peptide fragmentation in MS/MS analysis of specific peptide

29. Direct sequencing of peptides from target proteins by LC/MS/MS spectrometry

30. 6. Identification of pathway-involving proteins by specific Abs Phospho-specific antibodies ------ P-Tyr Ab P-Thr Ab etc. (Cell Signaling Tech.) ----Tyr---------- PO 4

31. Phospho-specific antibodies ------ P-Tyr Ab P-Thr Ab etc. ----Thr---------- PO 4

32. Nitro-Tyrosine specific antibodies --- NO pathway 3T3 C6 A431 HeLa 3T3 ----Tyr---------- NO

33. Acetylated-Lysine specific antibodies --- Acetylation pathway ----Lys-------- C2H5C2H5

34. Phospho-(Thr) MAPK/CDK Substrate Antibody --- MAPK/CDK pathway ----Thr-Pro------- PO 4