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Applications of antibody in biomedical research/proteomics

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. Cell cycle analysis of cultured newt lung cells. Structure of antibody. Classes of antibody.

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Applications of antibody in biomedical research/proteomics

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  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 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:

  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 Cell Extracts SDS-PAGE E Detection Blotting Membrane

  12. 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

  14. Anti-peptide Ab of human PAK2 (62 kDa) IP/WB using A431 cell extracts ppt sup ppt - + - + peptide + - 1-17 B E E 2nd Antibody-AP Biotin-strepavidin-AP

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

  16. Mouse mAb of human HSP 70 (70 kDa) Control Heat shock 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. Bead Bead Protein A/G Protein A/G In proteomic study 5. Identification of specific protein-protein interaction by IP C C B E B E D D cfg ppt b-ME/heating SDS-PAGE B IgG heavy chain C D IgG light chain E

  20. 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

  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) Solid probe Time of Flight m/z ~ time of flight

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

  24. Protein identification by database search using peptide mapping software

  25. ?

  26. MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEAHSRMVIPGGIDVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRDIGAIAQVHAE NGDIIAEEQQRILDLGITGP EGHVLSRPEEVEAEAVNRAI TIANQTNCPL YITKVMSKSSAEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTLIPEGTNGTEERMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLVIWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLHVTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPPVRNLHQSGFSLSGAQIDDNIPRRTTQRIVAPPGGRANITS 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 P-Tyr Ab P-Thr Ab etc. ----Tyr---------- Phospho-specific antibodies ------ PO4 (Cell Signaling Tech.)

  31. P-Tyr Ab P-Thr Ab etc. ----Thr---------- Phospho-specific antibodies ------ PO4

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

  33. Acetylated-Lysine specific antibodies --- Acetylation pathway ----Lys-------- C2H5

  34. Phospho-(Thr) MAPK/CDK Substrate Antibody --- MAPK/CDK pathway ----Thr-Pro------- PO4

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