DNA和蛋白质之间的相互作用

Competition for binding to the radiolabeled probe using unlabeled wildtype and mutated oligos
R. Voll 09/01
Nuclear extract of activated cells
Nuclear extract of activated cells with anti-p50 antibody
细胞的固定 染色体的断裂 免疫沉淀 Reverse cross-linking
DNA purification and PCR
unspecific binding cross-linking too much cross-linking degradation of protein two important steps not enough digestion nuclease digestion too much digestion protein detach from chromatin
Cisregulatory element
exon1
Cisregulatory element P Luciferase
7
8
9
TGAGCTGGGG A
GAAGCC
p66P p66P p66P pGL basic
400
800
1200
1600
2000
TGAGCTGGGG A
GAAGCC
Nrf2+ Nrf2+ vector+
R. Voll 09/01
Competition with Unlabeled Oligos
p50/p65 p50/p50 Unspecific
Free probe
GGG GAC TTT CCC
Wild type oligo
GGA GAC TTT CCC
Mutated oligo
Increasing amounts of unlabeled oligos containing the NF-B binding site or unlabeled oligos with a mutated binding site were added to the reaction mix prior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.
R. Voll 09/01
Preparation of Nuclear and Cytosolic Extracts
The procedure is carried out on ice rsp at 4°C and in the presence of protease (and phosphatase) inhibitors. 1. Swell cells in hypotonic lysis buffer 2. Add NP-40 and vortex to disrupt cytoplasmic membrane 3. Centrifuge to pellet nuclei 4. Carefully remove supernatant (contains cytosolic and membrane fraction) 4. Wash nuclear pellet once in lysis buffer 5. Add hypertonic extraction buffer to nuclear pellet 6. Agitate vigouresly for 30 minutes 7. Centrifuge at high speed 8. Remove nuclear extract, determine protein concentration and freeze on dry ice until EMSA is performed
DNA 和蛋白质的相互作用 EMSA和ChIP
Luciferase Assay
2
Luciferase Assay
质粒构建
细胞转染
24-48小时
Dual Luciferase 分析
3
转染效率的矫正 共转染：Firefly 和 Renilla
萤火虫(Photinus pyralis ) 萤光素酶和海肾
p66P* p66P p66P
400
800 1200 1600
10
Gene Regulation by Transcription Factors
Regulatory Region
Coding Sequence
R. Voll 09/01
Application:
Detection of DNA-binding factors/proteins
R. Voll 09/01
Labeling the Probe
A. T4 Polynucleotide Kinase
5’-AGT TGA GGG GAC TTT CCC AGG-3’ 3’-CA ACT CCC CTG AAA GGG TCC G-5’ + Adenosin-P-P-P (g-ATP) PNK
Binding motif
R. Voll 09/01
Annealing the Oligos
Heat up an equimolar mixture of the 2 oligos to 95°C and let them slowly cool down by turning off the heat block.
R. Voll 09/01
Reagents
Competitor DNA: poly (dI-dC) . poly (dI-dC) Competition of unspecific binding (e. g. histones)
Radiolabeled Probe:
Detection of DNA-binding proteins
(Renilla reniformis ) 萤光素酶
4
Promoter and cisregulatory elements
Cisregulatory element exon1
enhancer, silencer, insulator, CG island, MAR, DNA tethering fragment
Supershift
p50/p65 + anti-p50 Radioactive labelled oligonucleotide with NF-B - binding site (probe) and bound NF-B
p50/p65
Free probe
Radioactive labelled oligonucleotide with NF-B - binding site (probe)
false positive signle
Try to find the best crosslinking time for our proteins
Hela S3 cells were treated with 1% prewarmed formaldehyde-DMEM for 2mins, 4mins, 6mins, 8mins separately at 37 centidegrade
•Analysis of DNA sequences (e. g. promoter or enhancer regions) for their potential to bind specifically to proteins/nuclear extracts •Analysis of (sub-)cellular extracts for the presence of certain DNA-binding proteins (e. g. a transcription factor with a known recognition sequence)
5’-P-AGT TGA GGG GAC TTT CCC AGG-3’ 3’-CA ACT CCC CTG AAA GGG TCC G-P-5’
R. Voll 09/01
Removal of free radioactive material
Remove not incorporated isotop by Sephadex G50 column
R. Voll 09/01
Nrf2
-
+
cyclohexmide
染色体免疫沉淀
cross-linking of target proteins to their DNA binding sites
precipitated target proteinsDNA
immunoprecipitation with specific antibodies
Reaction Buffer
Binding conditions
R. Voll 09/01
源自文库
Analysis by non-Denaturing Polyacrylamide Gel Electrophoresis
R. Voll 09/01
Proof of Specificity
Supershift using antibodies against the DNA-binding protein
A double-stranded oligonucleotide containig a NF-B- binding site is labelled radioactive and incubated with a nuclear extract. During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift compared to the free probe.
R. Voll 09/01
Nuclear extract of non-activated cells
Nuclear extract of activated cells
EMSA: Principle
Radioaktively labeled oligonucleotide with NF-B - binding site (probe) and bound NF-B
NF-B
Free Probe
Radioactively labeled oligonucleotide with NF-B - binding site (probe)
A double-stranded oligonucleotide containig a NF-B- binding site is labeled with a radioactive isotope and incubated with a nuclear extract. During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift compared to the free probe.
Prepare cell extract
Collect nucleio-protein using gradient centrifugation
2mins
4mins
6mins
8mins
nucleio-protein
gradient centrifugation to purify nucleio-protein from different cross-linked cells
R. Voll 09/01
The Probe
Double stranded radiolabeled oligonucleotides containing a transcription factor binding site
AP-1 5’-GCT TGA TGA CTC AGC CGG AA C-3’ 3’-CGA ACT ACT GAG TCG GCC TT G-5’ NF-B 5’-AGT TGA GGG GAC TTT CCC AGG C-3’ 3’-TCA ACT CCC CTC AAA GGG TCC G-5’