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Protein

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Protein protein interaction: MIC-1 + + + + - wt wt wt mt : Ab ( -E2F1) - - - - + wt : probe (E2F) - - - - + E2F + IgG E2F ... – PowerPoint PPT presentation

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Title: Protein


1
Protein protein interaction
2
Modular Organization of Protein Interaction
Network
3
Biological Networks
Biological networks as framework for the study of
biological systems
4
A Section of Module Network of 30 Largest Modules
5
METHODS FOR SHOWING INTERACTIONS
  • Between protein and another protein
  • immunoprecipitation (in vivo)
  • GST pull-down assay (in vitro)
  • yeast two-hybrid system (in yeast)

6
GST pull down assay
7
GST pull down assay
Principle
a simple technique to test interaction between a
tagged protein or the bait (GST, His6, biotin
...) and another protein (test protein, or prey).
Method
8
1. Engineered protease site allows removal of
fusion partner
9
Sepharose GSH
10
Sepharose GSH
11
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12
Tracheae defective/Apontic is an MBF1 partner
13
2. Addition of a few residues should have minimal
effect on recombinant protein
  • His6 Tag
  • add 6 consecutive His to either end
  • binds metals
  • Epitope Tag
  • 6-12 amino acids
  • mAb for detection or purification

14
Immunoprecipitation
15
Immunoprecipitation
  • affinity purification based on isolation of Ag-Ab
    complexes
  • analyze by gel electrophoresis
  • initially based on centrifugation of large
    supramolecular complexes
  • high and ?equal amounts
  • isolation of Ag-Ab complexes
  • fixed S. aureus
  • protein A-agarose
  • protein G-agarose
  • Bacterial proteins that bind IgG (Fc)
  • protein A (Staphylococcus aureus)
  • protein G (Streptococcus)
  • binds more species and subclasses

16
Immunoprecipitation(????)
Method
Cell lysate ??
Cell lysate Preclearing
Immunoprecipitation?? ? ??? ? ? ?? chromosome??
Proteins A or Proteins G? ???? ??
antigen-antibody? ????? ??? ?? the bead? ?? ? ??
??? ???? ??
Immunoprecipitation
precleared lysate? ???? tube? 110ug? antibody?
?????
Washing Ab? ???? ??
???? ??? Washing buffer? ???? washing ?? ?
SDS-PAGE loading ? wastern blotting ?? ??
DNase foot printing method
Figure. Immunoprecipitation
17
Typical IP Protocol
  • 1. Solubilize antigen
  • usually non-denaturing
  • SDS excess of TX100
  • 2. Mix extract and Ab
  • 3. Add protein G-agarose, etc
  • 4. Extensively wash
  • 5. Elute with sample buffer
  • 6. SDS-PAGE
  • 7. Detection
  • protein stain
  • radioactivity

G
18
Tracheae defective/Apontic is an MBF1 partner
19
Example
MIC-1 (ng/ml)
50
0
20
Rb
IP Rb
E2F
CD9 co-immunoprecipitates with aIIb  3 in
Brij-35- (BRIJ), but not TX-100-solubilized
platelets
Detergent Strong TritonX-100, NP-40 Mild
Brij, CHAPS
20
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21
Fusion Proteins
  • increase stability
  • affinity purification
  • detection/assay
  • spectrophotometric
  • binding assays
  • antibodies
  • export signals

22
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23
Yeast two hybrid
24
The yeast two hybrid system
Principle
protein-protein interactions ? ?? ?? ?? ??
Eukaryote? ?? complex? ???? signal? ???? ???
??? ??? complex? ???? ???? protein? ?? ? ?? ????
?? protein? DNA binding domain? activation
domain ???? ??? binding ? ??? ???? protein? ?? ?
yeast? ?? ??? ?? protein? binding? ?? ?
Method
Figure.
Yeast two hybrid system
25
The yeast two hybrid system
26
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27
Yeast 2-Hybrid Assay
28
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29
Example
Figure. PIAS ? Smad ?? ???? ?? ??
Purpose PIAS? Smad6, 7? binding ??? ?? ? yeast
GAL4 DB - Smad7,6 MH2 yeast GAL4 AD - PIYS mm
medium ?? ? ??? ?? ?? ??
Result Smad 7? PIYS? ?? ??? yeast ?? ?? ??? ???
?? ??? ??. ? ? ???Binding ?? ? ? ??
JBC. Vol, 278, pp 34253- 34258 Swiyu Imoto,
Kenji Sugiyama and Ryuta Muromoto
30
Yeast two-hybrid assay
31
protein - DNA interaction
32
Gene expression? ? ????
Signal
Signal
??? ????? ????? ????? ?? ??? ????? ??
A cell
B cell
Behavior
Behavior
Behavior
33
Structure of gene and its promoter
34
rat GLUT2 gene structure (1995)
35
DEFINITION Human mRNA for p53 cellular tumor
antigen ACCESSION X02469 M60950 SOURCE human ORGA
NISM Homo sapiens AUTHORS Zakut-Houri,R.,
Bienz-Tadmor,B., Givol,D. and Oren,M. TITLE
Human p53 cellular tumor antigen cDNA sequence
and expression in COS cells JOURNAL EMBO J. 4
(5), 1251-1255 (1985) FEATURES
Location/Qualifiers source 1..1317
/organism"Homo sapiens"
/db_xref"taxon9606" CDS
136..1317
/note"p53 tumor antigen (aa 1-?)"
/codon_start1
/protein_id"CAA26306.1"
/db_xref"PIDg35210"
/db_xref"GI35210"
/db_xref"SWISS-PROTP04637"
/translation"MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLP
SQAMDDLM LSPDDIEQWFTEDPGPDEAP
RMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKT
YQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCP
VQLWVDSTPPPGTRVRAM
AIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRN
TFRHSVVV PYEPPEVGSDCTTIHYNYMC
NSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCA
CPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSP
QPKKKPLDGEYFTLQIRG
RERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLM
FKTEGPDSD"
36
CDS 136..1317 ORIGIN 1
gtctagagcc accgtccagg gagcaggtag ctgctgggct
ccggggacac tttgcgttcg 61 ggctgggagc
gtgctttcca cgacggtgac acgcttccct ggattggcag
ccagactgcc 121 ttccgggtca ctgccatgga
ggagccgcag tcagatccta gcgtcgagcc ccctctgagt
181 caggaaacat tttcagacct atggaaacta cttcctgaaa
acaacgttct gtcccccttg 241 ccgtcccaag
caatggatga tttgatgctg tccccggacg atattgaaca
atggttcact 301 gaagacccag gtccagatga
agctcccaga atgccagagg ctgctccccc cgtggcccct
361 gcaccagcag ctcctacacc ggcggcccct gcaccagccc
cctcctggcc cctgtcatct 421 tctgtccctt
cccagaaaac ctaccagggc agctacggtt tccgtctggg
cttcttgcat 481 tctgggacag ccaagtctgt
gacttgcacg tactcccctg ccctcaacaa gatgttttgc
541 caactggcca agacctgccc tgtgcagctg tgggttgatt
ccacaccccc gcccggcacc 601 cgcgtccgcg
ccatggccat ctacaagcag tcacagcaca tgacggaggt
tgtgaggcgc 661 tgcccccacc atgagcgctg
ctcagatagc gatggtctgg cccctcctca gcatcttatc
721 cgagtggaag gaaatttgcg tgtggagtat ttggatgaca
gaaacacttt tcgacatagt 781 gtggtggtgc
cctatgagcc gcctgaggtt ggctctgact gtaccaccat
ccactacaac 841 tacatgtgta acagttcctg
catgggcggc atgaaccgga ggcccatcct caccatcatc
901 acactggaag actccagtgg taatctactg ggacggaaca
gctttgaggt gcgtgtttgt 961 gcctgtcctg
ggagagaccg gcgcacagag gaagagaatc tccgcaagaa
aggggagcct 1021 caccacgagc tgcccccagg
gagcactaag cgagcactgc ccaacaacac cagctcctct
1081 ccccagccaa agaagaaacc actggatgga gaatatttca
cccttcagat ccgtgggcgt 1141 gagcgcttcg
agatgttccg agagctgaat gaggccttgg aactcaagga
tgcccaggct 1201 gggaaggagc caggggggag
cagggctcac tccagccacc tgaagtccaa aaagggtcag
1261 tctacctccc gccataaaaa actcatgttc aagacagaag
ggcctgactc agactga
37
ATG?
ATG?
5 end of 1st exon missing when cDNA cloning can
be determined by 1) Primer extension 2) RNase
protection assay (RNase mapping) 3) S1 nuclease
assay 4) 5-RACE (Rapid Amplification of cDNA
End)
38
Trancription machinery components
General (Basal) factors Upstream
factors ubiquitous not regulated initiation
efficiency ? Inducible factors similar to
upstream factors regulatory role at specific
time and specific tissue binding site is called
"response element"
Definition of promoter and enhancer Promoter
responsible for only initiation (200
bp) Enhancer enhance initiation, closely
packed array (100 bp)
39
Promoter Characterization It is not possible to
predict the DNA sequence recognized by proteins
1) Upstream factors
Basal factor (TATA, Inr) initiation
location Upstream factor (GC, CAAT) frequency of
initiation (assembly) Conserved element does not
inevitably imply binding of protein
40
2) Response elements
  May be located in promoters or enhancers Active
protein is available only under certain
condition Any one of several elements can
independently activate the gene
41
3) Character of transcription factors   Structure
? DNA-binding domain (usually basic) ?
Activation domain (usually acidic) ?
Dimerization domain Domains are independent,
interchangable
42
Transactivation direct interaction or with
coactivators contact with TFIID (most common,
esp TAFs), TFIIB, TFIIA can influence to
initiation complex by looping at a distance
43
Categories of Transcription Activators
according to DBD, Gene VII
1) Helix-turn-helix (HTH) motif 2) Zn finger
motif3) Leucine zipper (Zip) motif, usually
basic (bZip) 4) Helix-loop-helix (HLH) motif,
usually basic (bHLH)
1) Helix-Turn-Helix motif
two or three ? helices and short chain (turn) can
form dimer Homeodomain proteins found in
proteins related to development originally found
in Drosophila determine the identity of body
structure also found in higher eukaryote Oct
proteins 75 aa, called Pou domain
44
2) Zinc Finger motif
? helix - Zn - ? sheet ? helix contacts
DNA   Classic zinc finger proteins Sp1 (3
fingers) Steroid receptors 2 fingers steroid
hormone MR, AR, PR, ER thyroid hormone
T3R retinoic acid (vitamin A) RAR, RXR bind
to a specific receptor that activates gene
transcription "receptor" may be a
misnomer recognize special consensus sequence,
like GRE consist of central DNA binding
domain N-terminal activation domain C-terminal
ligand binding domain
45
Additional) b-barrels
two ? sheets contacts DNA  Papilloma virus
activator E2
3) Leucine Zipper motif
two ? helices DNA binding and protein
dimerization by same motif homodimer or
heterodimer expand the repertoire of DNA-binding
specificities basic region is DNA binding domain,
bZip   C/EBP Jun/Fos, JunB, JunD, Fra
(Fos-related Antigen) Fos cannot
homodimerize Jun/Fos can bind with an activity
more 10 folds than Jun/Jun
46
4) Helix-Loop-Helix motif
short ? helix and long ? helix homodimer or
heterodimer highly basic region can bind to DNA
bHLH   E12, E47 (Ig gene enhancer) MyoD,
myogenin, Myf-5 (myogenesis), Myc
(oncogene)   bHLH fall into 2 groups class A
ubiquitously expressed (E12/E47) class B
tissue-specific manner (MyoD) some HLH protein
lacks long helix can dimerize, unable to
bind dominant negative way (Id proteins)
47
dominant negative fashion like HLH proteins
48
PROMOTER ANALYSIS   1) Oocyte system 2)
Transfection system CAT assay Luciferase
assay 3) Transgenic system 4) in vitro
system EMSA (electrophoretic mobility shift
assay) DNase I footprinting assay In vitro
transcription assay 5) Transcription factor
characterization Affinity chromatography Two
hybrid 6) in vivo system in vivo DNase I
footprinting assay ChIP assay (chromatin
immunoprecipitation)
49
How to measure gene activation in eukaryotic
cells ?
Transfection assays
50
How to measure gene activation in eukaryotic
cells ?
Transfection assays
51
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52
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53
DNA foot printing
54
DNase foot printing
Principle
protein? DNA interaction? ?? ?? ?? ?? protein?
DNA? binding? ?? protein? ?? DNA? ?? ???? DNase
I? ???? ??? DNA? ??? ?? ? ?? gel loading ? DNA
band? ??? ?? ??? ?? ? ??? foot print? ?

55
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56
Fig. DNase foot printing
57
Method

?? protein binding site? ???? ??
Restriction fragment ?? ?? ??? ?? ??(??
32P??) ???? labeling ? ??? ????? ?? ? DNA? ??
?? ??? DNA Protein? ?? ???? ??? DNase I? ??
DNase I DNA?? single brake? ??? ?? ? ??
DNase I? mild?? ???? ?. ?? ??? strand ?
??? nick? ?? ? DNase I? ??? stop ??.
DNA? denatured ?? ??? denaturing polyacrylamide
gel running ?? ?? ?? running ?? DNA band?
autoradiography ?? Phosphorimager? ???? ?? ?
??? ??? band? ladder? DNasae I? ?? ?? ???
site ? ?? ? Control? ???? Band? ???? ?? ??? ??
Protein? DNA? binding ? ???? ??? sequence ?
size? ?? sequencing ??? ?? ? ? ??
58
Example
HA-NFATp binds DNA alone and cooperatively with
cJun/cFos. DNase I footprinting assays were
performed to investigate the binding of HA-NFATp
to a region of the human IL-2 promoter in the
absence (lanes 1-5) and presence (lanes 6-10) of
recombinant human cJun/cFos (3.2 nM). HA-NFATp
was added to reactions at the following final
concentrations 3 nM, lanes 2 and 7 9 nM, lanes
3 and 8 27 nM, lanes 4 and 9 and 54 nM, lanes 5
and 10. Footprinting reactions were resolved by
denaturing PAGE and analyzed with a Molecular
Dynamics PhosphorImager. Positions relative to
the transcriptional start site (1) of the human
IL-2 promoter are indicated on the left.
Locations of the previously characterized high
affinity -45 NFAT site and composite element are
indicated on the right.
59
EMSA(electrophoretic mobility gel shift assay )
Principle
Protein-DNA complex Protein-DNA complexes migrate
more slowly than free DNA molecules when
subjected to non-denaturing polyacrylamide or
agarose gel electrophoresis
60
Method
Figure.
EMSA
61
protocol
  • Nuclear extract preparation
  • Drug treatment
  • Washing 1 times with cold PBS (7ml)
  • Scrape with cold PBS 1ml
  • Centrifuge at3,000 rpm for 1min
  • Resuspend with buffer A 400ul
  • Incubate on ice for 15min
  • Add 25ul of 10 NP-400 (final conc 0.6)
  • Vortexing or pipetting for 30sec (???)
  • Centrifuge at 14,000rpm for 2-5min in 4C
  • Resuspend with buffer C 50ul
  • Incubate on ice for 30min
  • Centrifuge at 14,000rpm for 5min in 4C
  • Transfer 50ul of the soup to fresh tube
  • Storage at -70c in deep freezer

62
Buffer A -10mM HEPES pH 7.9, 10mM KCL, 0.1mM
EDTA, 0.1mM EGTA, 1mM DTT, 0.5mM PMSF, 0.5mM
Leupeptin Buffer C -20mM HEPES pH 7.9, 0.4M NACL
1mM EDTA, 1mM EGTA, 1mM DTT, 1mM PMSF, 1mM
Leupeptin
63
protocol
64
protocol
65
protocol
66
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67
Example
wt
wt
wt
mt
wt
probe (E2F)
Free probe
-
-
-
-

Cold probe
-
-
-
-
Ab (a-E2F1)

-




MIC-1
E2F IgG
E2F
Free probe
c-Jun
JunB
JunD
FosB
Fra-1
c-fos
Fra-2
-
-
-
-
Ab
Free probe
Probe
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
mt
-
-
-
-
-
-
-
-
-
-
Cold probe
wt
-









FN

AP-1 IgG

AP-1
68
ChIP(chromatin immunoprecipitation)
Principle
Protein ? ?? ??? ??? ?????? ???? ??
Protein? specific DNA region? ???? ?? ??? ??
protein Ab? immunoprecifitation?? ? primer? ??,
PCR? ???? ??
69
Chromatin immunoprecipitation
  • Used to determine whether a given protein binds
    to a given DNA sequence in vivo
  • Like all protein analysis involving antibodies
    (including westerns) a specific antibody is
    required
  • If there is no specific antibody, then epitope
    tagging can be employed (FLAG, MYC, HIS)
  • An epitope is a portion of a molecule to which an
    antibody binds

70
Method
???????? ???(DNA? ??? ???)? DNA? ??????.
  Sonication ??? ??? DNA? ?? ???. Immunoprecip
itation ?? ?? DNA? ?? ??   ??? ?? ??? ? ????
???? primer? ??? ?? PCR? ??.    ?? ?? ????? A??
?? ???? ????? ????? ?? ??? ??? ???? DNA? ???? A
??? ???? ??? ???, ??? A??? ????? ???? primer? ??,
PCR? ???? ??? ????. ??? ??? ??? ??? ???? ??? ???
??? ???? ???? ?
Figure.
Yeast two hybrid system
71
Chromatin ImmunoPrecipitation (ChIP)
- Purification of nuclei from 0-16 hrs embryos -
UV cross-linking
- Sonication (0.1 - 3 Kb)
- in vivo IP with anti-EN Ab
  • Addition of linkers
  • PCR amplification

- in vitro IP with anti-EN Ab
PCR amplification. HindIII digestion and cloning
in Ks 603 individual clones isolated and
sequenced
315 independent clones
72
Example
Snail promoter ???? MTA3? MBD3??? Association??
??.
MTA3? MBD3??? Snail promoter Start ??? 500bp ??
??? upstream ???? ??? Binding??.
73
ChIP analysis on estrogen target genes.
Mol. Cell Biol. (2004)
74
Knockdown of RIZ1 by siRNA affects expression
and methylation of pS2 gene.
Mol. Cell Biol. (2004)
75
protocol
  • Part A. Optimization of DNA ShearingEstablish
    optimal conditions required for shearing
    cross-linked DNA to 200-1000 base pairs in length
    by following steps 1- 9 below. Vary the power
    setting and/or the number of 10-second pulses
    during sonication of the samples. Be sure to keep
    the sample on ice at all times (the sonication
    generates heat which will denature the DNA).
    Check the size of sonicated DNA by gel
    electrophoresis after reversion of cross-links.
    Our experience shows DNA is sheared to the
    appropriate length with 3-4 sets of 10-second
    pulses using a Cole Parmer, High Intensity
    Ultrasonic Processor/Sonicator, 50 watt model
    equipped with a 2mm tip and set to 30 of maximum
    power. Once sonication conditions have been
    optimized, keep cell number consistent for
    subsequent experiments. The protocol below for
    the optimization of DNA Shearing is for one Chip
    assay (1 x 106 cells per condition).
  • Note Steps 3 - 7 should be done on ice
  • Stimulate or treat 1 x 106 cells on a 10cm dish
    as appropriate. (Cells should be treated
    under   conditions for which transcriptional
    activation of the gene of interest has been
    demonstrated). Include   one extra dish (1 x 106
    ) to be used solely for estimation of cell
    number.
  • 2. Cross link histones to DNA by adding
    formaldehyde directly to culture medium to a
    final concentration
  •    of 1 and incubate for 10 minutes at 37C. (For
    example, add 270 microliters 37 formaldehyde
    into
  • 10mof growth medium on plate).
  • 3. Aspirate medium, removing as much medium as
    possible. Wash cells twice using ice cold
  • PBS  containing protease inhibitors (1mM
    phenylmethylsulfonyl fluoride (PMSF),
    1microgram/ml
  • aprotinin and  1microgram/ml pepstatin A). Note
    Add protease inhibitors to PBS just prior to use.
  • PMSF has a half-life of approximately 30 minutes
    in aqueous solutions.
  • 4. Scrape cells into conical tube.

76
5. Pellet cells for 4 minutes at 2000 rpm at 4ºC.
Warm SDS Lysis Buffer (Catalog 20-163) to
room    temperature to dissolve precipitated SDS
and add protease inhibitors (inhibitors 1mM
PMSF,   1microgram/ml aprotinin and 1microgram/ml
pepstatin A). 6. Resuspend cell pellet in 200
microliters of SDS Lysis Buffer (Catalog
20-163) and incubate for 10    minutes on ice.
Note The 200 microliters of SDS Lysis Buffer is
per 1 X 106 cells if more cells are   used, the
resuspended cell pellet should be divided into
200 microliters aliquots so that each
200ml    aliquot contains 1 X 106 cells. 7.
Sonicate lysate to shear DNA to lengths between
200 and 1000 basepairs being sure to keep
samples  ice cold (Note Once sonication
conditions have been optimized following steps 1
to 9, proceed to  Part B, step 1 below).
8. Add 8 microliters 5M NaCl (Catalog 20-159)
and reverse crosslinks at 65ºC for 4 hours. 9.
Recover DNA by phenol/chloroform extraction and
run sample (example 5 microliter, 10 microliter,
  and 20 microliter samples) in an agarose gel
to visualize shearing efficiency.
77
protocol
  • Part B. Experimental protocol. If sonication
    conditions have been optimized (Part A), complete
    steps 1 through 7 and continue with the protocol
    below. For a negative/background control, prepare
    a sample to use as a no-antibody
    immunoprecipitation control in step 5 below.
    Additionally, transcriptionally unactivated DNA
    samples should be prepared as controls for PCR in
    section II.
  • Centrifuge samples (part A, step 7) for 10
    minutes at 13,000 rpm at 4C, and add 200
    microliters of the sonicated cell pellet
    suspension to a new 2ml-microcentrifuge tube.
  • 2. Dilute the sonicated cell pellet suspension 10
    fold in ChIP Dilution Buffer (Catalog 20-
  • 153), adding protease inhibitors as above. This
    is done by adding 1800 microliters ChIP
  • Dilution Buffer to the 200microliter sonicated
    cell pellet suspension for a final volume of 2ml
    in
  • each immunoprecipitation condition. Note If
    proceeding to PCR a portion of the diluted cell
  • pellet suspension 1 (20 microliters) can be
    kept to quantitate the amount of DNA present in
  • different samples at the PCR protocol, Part B,
    section II, step 6. This sample is considered to
  • be your input/starting material, andneeds to
    have the Histone-DNA crosslinks reversed by
  • heating at 65C for 4 hours (see section II,
    step3.)
  • 3. To reduce nonspecific background, pre-clear
    the 2ml diluted cell pellet suspension with 80
  • microlitersof Salmon Sperm DNA/Protein A
    Agarose-50 Slurry (Catalog 16-157) for 30
  • minutes at 4ºC withagitation.
  • 4. Pellet agarose by brief centrifugation and
    collect the supernatant fraction.

78
5. Add the immunoprecipitating antibody (the
amount will vary per antibody) to the 2ml
supernatant fraction and incubate overnight at
4ºC with rotation. For a negative control,
perform a no-antibody  immunoprecipitation by
incubating the supernatant fraction with 60
microliters of Salmon SpermDNA/Protein A
Agarose- 50 Slurry (Catalog 16-157) for one
hour at 4ºC with rotation and proceedto step
7. 6. Add 60 microliters of Salmon Sperm
DNA/Protein A Agarose Slurry (Catalog 16-157)
for one hour at4ºC with rotation to collect the
antibody/histone complex. 7. Pellet agarose by
gentle centrifugation (700 to 1000 rpm at 4ºC,
1min). Carefully remove thesupernatant that
contains unbound, non-specific DNA. Wash the
protein A agarose/antibody/histonecomplex for
3-5 minutes on a rotating platform with 1ml of
each of the buffers listed in the order asgiven
below a) Low Salt Immune Complex Wash Buffer
(Catalog 20-154), one wash b) High Salt Immune
Complex Wash Buffer (Catalog 20-155), one wash
c) LiCl Immune Complex Wash Buffer (Catalog
20-156), one wash d) 1X TE (Catalog 20-157),
two washes   After step 7 above, the sample is
now a protein A/antibody/histone/DNA complex
ready for either an Immunoprecipitation/Immunoblot
assay (Section I) or Polymerase Chain Reaction
(PCR) assay (Section II)
79
protocol
Section I. Immunoprecipitation/Immunoblot
protocol to detect histone. 1 Following washing
of the beads in part B, step 7,
immunoprecipitated histones can be analyzed
by  immunoblot analysis. Add 25 microliters of 1X
Laemmli buffer per sample and boil for 10
minutes. Load  20 microliters per lane and
perform immunoblot procedure as described per
appropriate antibody. Section II. PCR protocol
to amplify DNA that is bound to the
immunoprecipitated histone. 1. Freshly prepare
elution buffer (1SDS, 0.1M NaHCO3). 2. Elute the
histone complex from the antibody by adding 250
microliter elution buffer to the pelleted protein
A agarose/antibody/histone complex from step 7d
above. Vortex briefly to mix and incubate at room
temperature for 15 minutes with rotation. Spin
down agarose, and carefully transfer
the  supernatant fraction (eluate) to another
tube and repeat elution. Combine eluates (total
volume approximately 500 microliters.) 3. Add 20
microliters 5M NaCl (Catalog 20-159) to the
combined eluates (500 microliters) and
reverse  histone-DNA crosslinks by heating at
65ºC for 4 hours. At this step the sample can be
stored and -20C and the protocol continued the
next day.
80
Note Include the input/starting material (the
sample saved from Part B, step 2, which has had
the Histone-DNA crosslinks reversed) as well as a
transcriptionally-unactivated DNA sample as
negative and background controls for the PCR
reaction. Previously, a 5 microliter sample has
been used in a nested PCR reaction. However, the
amount of sample used per reaction must be
determined empirically (e.g., titrate the sample
at this step by using 1, 2, 5, or 10 microliters
per PCR reaction). If PCR results are poor,
complete steps 4, 5 and 6 below to purify the DNA
sample. NOTE Handle the samples carefully, some
DNA may be lost during the purification
steps. 4. Add 10 microliters of 0.5M EDTA
(Catalog 20-158), 20 microliters 1M Tris-HCl,
pH 6.5 (Catalog  20-160) and 2 microliters of
10mg/ml Proteinase K to the combined eluates and
incubate for one hour at 45ºC. 5. Recover DNA by
phenol/chloroform extraction and ethanol
precipitation. Addition of an inert carrier,such
as 20 micrograms glycogen or yeast tRNA, helps
visualize the DNA pellet. Wash pellets with
70ethanol and air dry. 6. Resuspend pellets in
an appropriate buffer for PCR or slot-blot
reactions. PCR or slot-blot conditionsmust be
determined empirically.
81
ChIP on ChipChromatin immunopreciptation on a
microarray Chip
DNA from binding site
Fluorescently label
http//www.chiponchip.org/
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