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Electrophoretic Mobility S

hift Assay (EMSA)

Dr. Songyot Anuchapreeda


Department of Clinical Microscopy
Faculty of Associated Medical Sciences
Chiang Mai University
5% polyacrylamide gel
-198
5’CTAGAGAGGTGCAACGGAAGCCAGAACATTCCTCC
TGGAAATTCAACCTGTTTCGCAGTTTCTCGAGGAATC
AP-1 like motif
AGCATTCAGTCAATCCGGGCCGGGAGCAGTCATCTGT
Y box SP1
GGTGAGGCTGATTGGCTGGGCAGGAACAGCGCCGGGG
SP1 SP1
CGTGGGCTGAGCACAGCCGCTTCGCTCTCTTTGCCAC
AGGAAGCCTGAGCTCATTCGAGTAGCGGCTCTTCCAA
+1
GCTCAAAGAAGCAGAGGCCGC 3’
+43
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 37C for 45 min and then stop reaction with 1L of
0.5 M EDTA then add 15 L of DEPC treated water.
Method used in labeling nucleoti
des probe
 End labeling
 Polymerase-based labeling
 Nick translation of DNA
ATP
End-labeling probe DNA

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/L
Blank 47.8
59.3
Reference 14,586,732.2
Before Chroma spin column 515,764.3
After Chroma spin column
188,179.4
Labeled 32P
Oligonucleotide 1 2 3 4 5 6

Origin
1 = SP1
2 = AP1
3 = TFIID
4 = NFkB
5 = Oct
6 = CREB

KB-V1 nuclear extract

Free probe
No competitor Competitor

Nuclear protein,
50 mg
Protein binds to
unlabelled
competitor

Gel electrophoresis

DNA binding protein

Free probe

Autoradiography
Competiters
(cold probe)
KB-V1
P3P4
Curcumin treated cells (KB-V1)

Cont SP1 AP1 AP2 Oct CREB NFkB TFIID Cont SP1 AP1 AP2 Oct CREB NFkB TFIID
CREB consensus sequence
Cold CREB Cold probe 50 molars excess
Competitors
- + Cont SP1 AP1 AP2 TFIID NFkB Oct CREB

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
P3P4

1 2 3 4
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

Origin

Protein - DNA complex

Note
C = Control
2p = Isopropanol step
B = Bisdemethoxycurcumin
Sig = Sigma
Bk = Bisdemethoxycurcumin
from Kalsec
No supershift Supershift
Ab

Nuclear protein,
50 mg
Protein binds to
unlabelled
competitor

Gel electrophoresis

Supershift

Free probe

Autoradiography
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
1 2 3 4 5
6 7 Origin

Free probe

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
1 2

Supershift

1 = control
2 = NE+Anti-CREB

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