1 / 29

Patrick Bruss Erin Shaneyfelt 2 May 2002

Detection of Epstein-Barr Nuclear Antigen-1 in HeLa Cells Using Electrophoretic Mobility Shift Assay. Patrick Bruss Erin Shaneyfelt 2 May 2002. Crystal Structure of EBNA-1 1. Native state is a dimer 8-stranded anitparallel beta barrel. EBNA-1 Bound to DNA 1. Blue= central core of protein

Download Presentation

Patrick Bruss Erin Shaneyfelt 2 May 2002

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Detection of Epstein-Barr Nuclear Antigen-1 in HeLa Cells Using Electrophoretic Mobility Shift Assay Patrick Bruss Erin Shaneyfelt 2 May 2002

  2. Crystal Structure of EBNA-11 Native state is a dimer 8-stranded anitparallel beta barrel

  3. EBNA-1 Bound to DNA1 • Blue= central core of protein • Gold=adjacent alpha helicies which also contact DNA • Binding site2= 5’-…TAGCATATGCTA…-3’ 3’-…ATCGTATACGAT…-5’

  4. Binding in vivo1 • two dimers shown • DNA sites separated by 3bp • same distance between EBNA-1 binding sites of oriP • proteins overlap and therefore conformational change in either protein, DNA or both • EBNA-1 is very rigid • DNA bends, thought to be crucial for DNA replication initiation

  5. Life Cycle of Epstein-Barr Virus3

  6. EBV Associated Proteins3 • after infection, 6 nuclear antigens are expressed • EBNA-1 maintains viral plasmid during latency and activates replication during the lytic cycle • EBNA-2 involved in immortalization of lymphocytes • EBNA-3(a-c) involved in transformation of human -lymphocytes • most information about mechanism is still unknown • EBNA-1 is the only proteins expressed in ALL EBV infected cells.

  7. Human Diseases Identified with EBV4 • Mononucleosis • Polyclonal B Lymphoproliferative Disease (PLD) • Burkitt’s Lymphoma • Nasopharyngeal Carcinoma (NPC) • Hodgkin’s Disease (HD)

  8. Henrietta Lacks (HeLa) Henrietta Lacks (HeLa) who died in 1951 from cancer of the cervix5

  9. Electrophoretic Mobility Shift Assay (EMSA)6 • apparent molecular weight of DNA-protein complex > unbound DNA • apparent mw of DNA-protein-antibody complex > DNA-protein complex > unbound DNA • used to identify DNA binding proteins

  10. Importance7 • studies have shown that major control of genes and gene expression is done through DNA-protein interactions • e.g.: DNA replication, recombination and repair, transcription, RNA processing, viral assembly • to understand function of interactions, need to know information about structure of DNA-protein complexes, thermodynamics, and kinetics • Eletrophoresis mobility shift assay (EMSA) developed by Garner and Revzin to study these characteristics8.

  11. Conditions of Binding7 • Protein-DNA complexes can be formed by mixing small amounts of protein with labeled DNA in low salt buffer • Formation of complexes can be influenced by many parameters • monovalent ion concentration-low ionic strength (<150mM) increase stability of interaction • presence of non-ionic detergents or carrier proteins-can stabilize product • time and temperature of binding reaction • protein concentration • type and concentration of competitor DNA • Nonspecific competitor such as poly(dA-dT) or poly(dI-dC) used to distinguish between specific and nonspecific binding

  12. Conditions of Native Polyacrylamide Gel7 • mobility of complexes determined by size, charge, and confirmation of protein bound to DNA • composition of gel and electophoretic conditions can alter mobility and stability • higher conc. polyacrylamide stabilizes complex

  13. Applications of EMSA Analysis7 • Quantification • stoichiometric relationships between different complexes • Specificity of protein • DNA binding- perform experiment in presence of increasing amounts of unlabelled competitor • if competitor has high affinity binding site, will compete and decrease visible concentration of detectable complex • Equilibrium constants • obtained by mixing known amount of labeled DNA with increasing conc. of protein and construct standard binding curve • point at which 50% labeled DNA is bound with protein =Keq • Conformational changes of DNA • bent DNA molecules migrate slower than linear (also if bend is in center=slower than on end) • by creating DNA fragments which alter placement of DNA binding site, can study bending activity if protein • Stoichiometric analysis • number of protein that bind per DNA fragment • e.g.- using two different-sized derivatives of same protein-complexes will form three bands (two=homodimers of each derivative + one=heterodimer)

  14. Pros/Cons of EMSA7 • Advantages • don’t need highly purified proteins • can resolve complexes that differ in protein and nucleic acid stoichiometry and/or conformation • easy to separate different species • Disadvantages • no information about sequence of binding site • difficult to adjust all parameters for complete optimization

  15. Procedure • Followed protocol in Peirce EMSA handout2 • Binding Reaction • components: (total volume=20uL) • nuclease free water • 10X Binding Buffer (Tris, KCL, DTT, pH 7.5) • 50% Glycerol • MgCl2 • Poly (dI•dC) (in Tris, EDTA, pH 7.5) • 1% NP-40 • DNA (biotinylated or not) • Protein/lysate • sometimes antibody9 • incubate 20min at room temp • add loading buffer

  16. 6% Polyacrylamide gel • 0.5X TBE + 40% acrylamide + APS + TEMED • polymerize 1hr+ • Load/Run gel • use 0.5X TBE buffer • ~200V, 20-25mA, about 20min. • Transfer to (+)nylon membrane • 0.5X TBE, ice cooled • 380mA • 30min. • UV crosslink (5min.)

  17. Block/Wash • Lightshift Blocking Buffer • Lightshift Stabilized Streptavidin-Horseradish Peroxide Conjugate (filtered) • Lightshift 1X Wash Buffer • Lightshift Equilibration Buffer • Detection • Lighshift Luminol/Enhancer Solution • Lightshift Stable Peroxidase Solution • measure chemiluminescene by cooled CCD camera • 5-15min. exposure

  18. Control reaction 1= EBNA control DNA 2=(1)+ EBNA extract 3= (1,2)+ unlabelled EBNA control DNA • Loading dye was omitted from lanes 1 and 2 and therefore they did not have enough glycerol and the DNA diffused away • Still see expected shift in lane 2 due to EBNA DNA-protein complex 1 2 3 experimental results expected results

  19. Control + HeLa cells 1= EBNA control DNA 2=(1)+ EBNA extract 3= (1,2)+ unlabelled EBNA control DNA 4=(1)+ Active Motif HeLa 5=(1)+ Dr. Mascotti HeLa • gel did not run correctly due to buffer dilution error • stopped immediately after lanes entered gel • no shift for EBNA control (lane2) • binding to site for both HeLa samples • binding of approximately same size protein 5 4 3 2 1

  20. Control + HeLa cells (#2) 1= EBNA control DNA 2=(1)+ EBNA extract 3= (1,2)+ unlabelled EBNA control DNA 4=(1)+ Active Motif HeLa 5=(1)+ Dr. Mascotti HeLa • shift in HeLa’s about same size as EBNA shift • could indicate presence of EBNA • or another protein of similar size that recognizes binding site • upper bands= nonspecific binding 1 2 3 4 5

  21. Control + HeLa [DNA] 1= EBNA control DNA 2=(1)+ EBNA extract 3= (1,2)+ unlabelled EBNA control DNA 4=(1)+ Active Motif HeLa 5=(1)+ Dr. Mascotti HeLa • EBNA control shift is missing • could be due to less template available to bind • forgot to put in reaction • Bands in HeLa lanes are the ones that match EBNA shift • most intense bands from previous gel • other bands are gone due to lower concentration of protein 5 4 3 2 1

  22. Preliminary conclusions • EMSA works correctly and detects DNA to at least 1femtomole • Found a few proteins in HeLa cells that recognize EBNA binding site • One of these proteins in each HeLa sample matches shift of EBNA protein-DNA complex • Could be specific or unspecific binding • Could be EBNA or a different protein that happens to have a similar size

  23. Electrophoretic Mobility Shift Assay (EMSA)6 • apparent molecular weight of DNA-protein complex > unbound DNA • apparent mw of DNA-protein-antibody complex > DNA-protein complex > unbound DNA • used to identify DNA binding proteins

  24. Nonspecific Binding and Antibody • none of the bands are as intense as expected based on other labs • other photons make extra spots that don’t have DNA • pockets of substrate between the membrane and saran wrap • contamination that could have gotten some DNA and substrate bound • not sure about the shifts or lanes present to get reliable results

  25. 1= biotin-control DNA 2= (1) + extract EBNA 3= (1,2) + unlabeled DNA 4= (1) + Active Motif 5= (1,4) + unlabeled DNA 6= (1,4) + antibody 7= (1) + Dr. M. HeLa 8= (1,7) + unlabeled DNA 9= (1,7) + antibody 10= (1,2) + antibody (control) Possible Interpretation #1 • lane 3- contamination ? (should not have a shift) • bottom of dye front ran off gel • intensities of HeLa inverted from other trials • lane 5- could be a bit of chasing- would indicate specific binding (but not the band that matches EBNA shift) • lane 6- could be a little higher, but is smeared • lanes 9,10- didn’t seem to work at all- low intensity indicates DNA loss

  26. 1= biotin-control DNA 2= (1) + extract EBNA 3= (1,2) + unlabeled DNA 4= (1) + Active Motif 5= (1,4) + unlabeled DNA 6= (1,4) + antibody 7= (1) + Dr. M. HeLa 8= (1,7) + unlabeled DNA 9= (1,7) + antibody 10= (1,2) + antibody (control) Possible Interpretation #2 • only see one shift from extracts • unlabeled DNA control still did not work • lane 10- could be supershift from antibody • lane 8,9- still did not work 1 2 3 4 5 6 7 8 9 10

  27. Conclusions • None of the earlier conclusions were disputed • there is binding in the HeLa lysates that match shift with EBNA • could be specific or nonspecific • Antibody could be binding and there is no change in shift due to charge interactions, or conformational changes that counteract the additional weight • Need to run the last experiment again to get reliable results

  28. References • 1. “Crystal Structure of EBNA-1.” Department of Microbiology and Immunology, University of Rochester Medical Center. www.urmc.rochester.edu/smd/mbi/grad2/herp99BB6.html. 2002. (25 April 2002). • 2. “Lightshift Chemiluminescent EMSA Kit.” Pierce. Rockford, IL, 2002. • 3. Solomon, Julie, Carla Fowler, and G. Cooper. “Epstein-Barr Virus.” www.brown.edu/Courses/Bio_160/Projects2000/Herpes/EBV/Epstein-Barr.html. Brown University, 2002. (25 April 2002). • 4. Kang, Myung-Soo, Ciu Chun Hang and Elliot Kieff. “Epstein-Barr virus nuclear antigen 1 activiates transcription from episomal but not integrated DNA and does not alter lymphocyte growth.” Proceedings of the National Academy of Sciences, USA. 98(6), 15233-15238, 2001. • 5. Potier, Beth. Harvard University Gazette. www.news.harvard.edu/gazette/2001/07.19/04-filmmaker.html. President and Fellows of Harvard College, 2002. (25 April 2002). • 6. Lissemore, J. “EMSA.” Molecular Genetics (BL465), John Carroll University, 24 April, 2002. • 7. Norman, Cecilia. “Electrophoresis mobility shift assay (EMSA).” SLU, Uppsala. www.plantae.lu.se/fskolan/arabidopsistexter/CeciliaNorman.html, (30 April 2002). • 8. Garner, M M. Rezvin, A. (1981) Nucl. Acids Res., 9 (13), 3047-3060 • 9. “Mouse Anti-Epstein Barr Virus Nuclear Antigen (EBNA-1) Monoclonal Antibody.” Chemicon International. CA, 2002.

  29. Acknowledgements • Dr. Mascotti • Dr. Lissemore • Pierce • Chemicon International

More Related