Post on 18-Nov-2014
transcript
Electrophoretic Mobility Shift Assay (EMSA)
Dr. Songyot AnuchapreedaDepartment of Clinical Microscopy
Faculty of Associated Medical SciencesChiang Mai University
5% polyacrylamide gel
5’CTAGAGAGGTGCAACGGAAGCCAGAACATTCCTCC
TGGAAATTCAACCTGTTTCGCAGTTTCTCGAGGAATC
AGCATTCAGTCAATCCGGGCCGGGAGCAGTCATCTGT
GGTGAGGCTGATTGGCTGGGCAGGAACAGCGCCGGGG
CGTGGGCTGAGCACAGCCGCTTCGCTCTCTTTGCCAC
AGGAAGCCTGAGCTCATTCGAGTAGCGGCTCTTCCAA
GCTCAAAGAAGCAGAGGCCGC 3’
-198
+43
+1
AP-1 like motif
SP1Y box
SP1 SP1
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
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.
Method used in labeling nucleotides probe
End labelingPolymerase-based labelingNick translation of DNA
ATP
End-labeling probe DNA
P
P
P
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.
Component for loading sample for competitive EMSA
Gel shift binding buffer 2 L
Nuclear extract 50 g/ reaction
Unlabeled oligonucleotide 1 L
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)
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
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
Autoradiography
Gel electrophoresis
Protein binds to unlabelled competitor
No competitor Competitor
DNA binding protein
Free probe
Nuclear protein, 50 mg
Competiters (cold probe)
P3P4
Cont SP1 AP1 AP2 Oct CREB NFkB TFIID Cont SP1 AP1 AP2 Oct CREB NFkB TFIID
KB-V1 Curcumin treated cells (KB-V1)
CREB consensus sequenceCold CREB
- +
Cont SP1 AP1 AP2 TFIID NFkB Oct CREB Competitors
Cold probe 50 molars excess
KB-V1 nuclear protein
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
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
C 2p B C C Sig Bk
Protein - DNA complex
Origin
NoteC = Control2p = Isopropanol stepB = BisdemethoxycurcuminSig = SigmaBk = Bisdemethoxycurcumin from Kalsec
Autoradiography
Gel electrophoresis
Protein binds to unlabelled competitor
No supershift Supershift
Supershift
Free probe
Nuclear protein, 50 mg
Ab
Supershift
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
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
1 2
1 = control
2 = NE+Anti-CREB
Supershift