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Electrophoretic Mobility Shift Assay (EMSA)
Dr. Songyot AnuchapreedaDepartment of Clinical Microscopy
Faculty of Associated Medical SciencesChiang Mai University
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5% polyacrylamide gel
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5’CTAGAGAGGTGCAACGGAAGCCAGAACATTCCTCC
TGGAAATTCAACCTGTTTCGCAGTTTCTCGAGGAATC
AGCATTCAGTCAATCCGGGCCGGGAGCAGTCATCTGT
GGTGAGGCTGATTGGCTGGGCAGGAACAGCGCCGGGG
CGTGGGCTGAGCACAGCCGCTTCGCTCTCTTTGCCAC
AGGAAGCCTGAGCTCATTCGAGTAGCGGCTCTTCCAA
GCTCAAAGAAGCAGAGGCCGC 3’
-198
+43
+1
AP-1 like motif
SP1Y box
SP1 SP1
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5% acrylamide gel (native gel)
10X TBE 2 mL
29:1 acrylamide/bis (W/W)(in 0.5XTBE 100 mL)6.7 mL
(Final = 30%)
Sterile DEPC treated water 31 mL
10% APS 167 L
205
TEMED 23 L
Total 40 m
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Phosphorylation reaction (for consensus oligonucleotide)
Consensus oligonucleotide ( 1.75 pmol/L) 2 L
T4 polynucleotide kinase (10X buffer) 1 L
[-32P] ATP (3,000 Ci/mmol at 10 mCi/ mL) 1 L
Nuclease free water 5 L
T4 nucleotide kinase (5-10 U/L) 1 L
Total volume 10 L
Incubate at 37C for 45 min and then stop reaction with 1L of
0.5 M EDTA then add 15 L of DEPC treated water.
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Method used in labeling nucleotides probe
End labelingPolymerase-based labelingNick translation of DNA
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ATP
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End-labeling probe DNA
P
P
P
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Component for loading sample
Gel shift binding buffer 2 L
Nuclear extract 50 g/ reaction
Labeled probe 1 L
Nuclease free water up to 10 L
Incubate the reactions at room temperature for 10 min, then
added 1 L of labeled probe. Incubate the reaction on ice
for 60 min. Added 1 L of gel loading 10X buffer per
reaction and analyze the products.
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Component for loading sample for competitive EMSA
Gel shift binding buffer 2 L
Nuclear extract 50 g/ reaction
Unlabeled oligonucleotide 1 L
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Percent incorporation and specific activity
Percent incorporation (%P) = After column (cpm) x 100
Before column (cpm)
Specific radioactivity (SA) = (Ci)(2.2 x 109)(P)
MI + [(1.3 x 103)(P)(Ci/SA)
Ci = Amount of radiolabeled nucleotide in microcuries in the
reaction mixture.
P = Proportion of radiolabeled nucleotide incorporated into
the probe DNA.
MI = Mass of input of the DNA template in ng.
SA = Specific activity of radiolabeled nucleotide in curies per
milimole (Ci/ mmol)
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Example: data after -counter are as follow:
Treatments CPM/LBlank
ReferenceBefore Chroma spin columnAfter Chroma spin column
47.859.3
14,586,732.2515,764.3
188,179.4
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1 2 3 4 5 6
Labeled 32P Oligonucleotide
1 = SP1
2 = AP1
3 = TFIID
4 = NFkB
5 = Oct
6 = CREB
Origin
Free probe
KB-V1 nuclear extract
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Autoradiography
Gel electrophoresis
Protein binds to unlabelled competitor
No competitor Competitor
DNA binding protein
Free probe
Nuclear protein, 50 mg
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Competiters (cold probe)
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P3P4
Cont SP1 AP1 AP2 Oct CREB NFkB TFIID Cont SP1 AP1 AP2 Oct CREB NFkB TFIID
KB-V1 Curcumin treated cells (KB-V1)
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CREB consensus sequenceCold CREB
- +
Cont SP1 AP1 AP2 TFIID NFkB Oct CREB Competitors
Cold probe 50 molars excess
KB-V1 nuclear protein
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1 2 3 4 origin
1 = Verapamil treated cells
(KB-V1).
2 = KB-3-1 (untreated cells)
3 = Curcumin treated cells
(KB-V1).
4 = KB-V1 (untreated cells)
Free probe
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1 2 3 4
P3P4
Origin
1 = KB-V1 (untreated cells)
2 = KB-3-1 (untreated cells)
3 = Curcumin treated cells
(KB-V1).
4 = Verapamil treated cells
(KB-V1).
Free probe
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C 2p B C C Sig Bk
Protein - DNA complex
Origin
NoteC = Control2p = Isopropanol stepB = BisdemethoxycurcuminSig = SigmaBk = Bisdemethoxycurcumin from Kalsec
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Autoradiography
Gel electrophoresis
Protein binds to unlabelled competitor
No supershift Supershift
Supershift
Free probe
Nuclear protein, 50 mg
Ab
Supershift
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Component for loading sample for Supershift assay
Anti-CREB 2 g
Gel shift binding buffer 2 L
Nuclear extract 50 g/
reaction
Labeled probe 1 L
Nuclease free water up to 10 L
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Origin
Lane 1. AP2 consensus oligonucleotide 5. NF-B consensus oligonucleotide 2. SP1 consensus oligonucleotide 6. OCT consensus oligonucleotide 3. AP1 consensus oligonucleotide 7. CREB consensus oligonucleotide 4. TFIID consensus oligonucleotide
Free probe
1 2 3 4 5 6 7
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1 2
1 = control
2 = NE+Anti-CREB
Supershift