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