Title: Detection of Epstein-Barr Nuclear Antigen-1 in HeLa Cells Using Electrophoretic Mobility Shift Assay
1Detection of Epstein-Barr Nuclear Antigen-1 in
HeLa Cells Using Electrophoretic Mobility Shift
Assay
- Patrick Bruss
- Erin Shaneyfelt
- 2 May 2002
2Crystal Structure of EBNA-11
- Native state is a dimer
- 8-stranded anitparallel beta barrel
3EBNA-1 Bound to DNA1
- Blue central core of protein
- Goldadjacent alpha helicies which also contact
DNA - Binding site2
5-TAGCATATGCTA-3
3-ATCGTATACGAT-5
4Binding 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
5Life Cycle of Epstein-Barr Virus3
6EBV 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.
7Human Diseases Identified with EBV4
- Mononucleosis
- Polyclonal B Lymphoproliferative Disease (PLD)
- Burkitts Lymphoma
- Nasopharyngeal Carcinoma (NPC)
- Hodgkins Disease (HD)
8Henrietta Lacks (HeLa)
Henrietta Lacks (HeLa) who died in 1951 from
cancer of the cervix5
9Electrophoretic Mobility Shift Assay (EMSA)6
- apparent molecular weight of DNA-protein complex
gt unbound DNA - apparent mw of DNA-protein-antibody complex gt
DNA-protein complex gt unbound DNA - used to identify DNA binding proteins
10Importance7
- 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.
11Conditions 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
(lt150mM) 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
12Conditions 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
13Applications 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 centerslower 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
(twohomodimers of each derivative
oneheterodimer)
14Pros/Cons of EMSA7
- Advantages
- dont 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
15Procedure
- Followed protocol in Peirce EMSA handout2
- Binding Reaction
- components (total volume20uL)
- nuclease free water
- 10X Binding Buffer (Tris, KCL, DTT, pH 7.5)
- 50 Glycerol
- MgCl2
- Poly (dIdC) (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
18Control reaction
1 EBNA control DNA 2(1) EBNA extract 3 (1,2)
unlabelled EBNA control DNA
1 2 3
experimental results
expected results
- 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
19Control 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
5 4 3 2 1
- 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
20Control 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
1 2 3 4 5
- shift in HeLas 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
21Control 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
5 4 3 2 1
- 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
22Preliminary 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
23Electrophoretic Mobility Shift Assay (EMSA)6
- apparent molecular weight of DNA-protein complex
gt unbound DNA - apparent mw of DNA-protein-antibody complex gt
DNA-protein complex gt unbound DNA - used to identify DNA binding proteins
24Nonspecific Binding and Antibody
- none of the bands are as intense as expected
based on other labs - other photons make extra spots that dont 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
25Possible Interpretation 1
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)
- 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- didnt seem to work at all- low
intensity indicates DNA loss
26Possible Interpretation 2
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)
1 2 3 4 5 6 7 8 9 10
- 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
27Conclusions
- 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
28References
- 1. Crystal Structure of EBNA-1. Department of
Microbiology and Immunology, University of
Rochester Medical Center. www.urmc.rochester.edu/s
md/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_16
0/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-filmmak
er.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/Cecili
aNorman.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.
29Acknowledgements
- Dr. Mascotti
- Dr. Lissemore
- Pierce
- Chemicon International