EMSA (Electrophoretic Mobility Shift Assay)

EMSA

Electrophoretic Mobility Shift Assay

Central question of EMSA

Does protein X bind to DNA Y (radiolabeled)?

What EMSA detects

DNA–protein interactions

How EMSA detects interactions

By observing changes in electrophoretic mobility on a gel

Key idea of EMSA

DNA–protein complexes migrate more slowly than free DNA

Which migrates faster

Free DNA migrates faster than DNA bound to protein

Where samples are loaded

At wells at the top of the gel

Effect of increasing protein concentration

More DNA appears shifted upward (slower migration)

What we visualize in EMSA

Radiolabeled DNA, not protein

Why only DNA is seen

Because DNA is radiolabeled, while protein is not

Bands for DNA–protein complexes

Higher, slower-moving bands

Bands for free DNA

Lower, faster-moving bands

Why DNA–protein bands move slowly

Complex is larger and migrates more slowly

Why free DNA bands move quickly

Smaller and migrates faster through the gel

What happens if multiple proteins bind

A “super shift” occurs (slower movement)

Definition of a super shift

Slower migration caused by multiple proteins and/or antibody bound to DNA

Case 1 of super shift

Two proteins bind at different DNA sites

Case 2 of super shift

One protein binds DNA, another protein binds the first protein

Effect of super shift

Even slower movement than DNA–protein complexes

Input DNA alone

Produces a fast-moving band at the bottom

Protein + input DNA

Produces two bands: DNA–protein complex and free DNA

Competitor DNA

Another DNA added to compete with input DNA for protein binding

Two characteristics of competitor DNA

(1) Cold (unlabeled), (2) Added in excess

Why competitor DNA is in excess

Increases probability of protein binding to competitor instead of input DNA

Specific competitor definition

DNA sequence that the protein can bind

Effect of specific competitor

Protein binds competitor DNA more than input DNA

Bands visible with specific competitor

Only free input DNA (competitor DNA is unlabeled)

Non-specific competitor definition

DNA sequence the protein cannot bind

Effect of non-specific competitor

Protein still binds input DNA

Bands visible with non-specific competitor

1. Input DNA alone

2. input DNA–protein complex

Antibody role in EMSA

Verifies that the DNA shift is due to the protein of interest

Effect of antibody binding

Antibody binds protein, causing a super shift

Bands possible with antibody

1. Free DNA

2. DNA–protein complex

3. DNA–protein–antibody super shift

Why not all antibody bands appear

Depends on protein/antibody concentrations and affinities

If antibody is in excess

DNA–protein band may disappear (all complexes super-shifted)

What EMSA autoradiogram shows

Only radiolabeled DNA species

Competitor DNAs on autoradiogram

Not visible (unlabeled)

Lane with input DNA only

Single fast-moving DNA band

Lane with protein + input DNA

DNA alone band + DNA–protein complex band

Lane with protein + specific competitor + input DNA

Mostly free input DNA band

Lane with protein + non-specific competitor + input DNA

Free DNA and DNA–protein complex

Lane with protein + antibody + input DNA

Free DNA, DNA–protein, and super-shifted DNA–protein–antibody

Why radiolabeling is essential

Ensures only input DNA is visualized

Reason for slower migration of complexes

Larger molecular complexes migrate more slowly

Reason for competitor DNA excess

Outcompetes labeled DNA probabilistically

Limitation of EMSA with multiple proteins

Cannot distinguish which protein binds DNA

Four indistinguishable EMSA cases

Protein A + DNA;

Protein B + DNA;

Protein A + Protein B + DNA;

Protein A–Protein B + DNA

How to confirm which protein binds DNA

Add an antibody specific to that protein

Technique to map DNA binding sequences

DNA footprinting

Role of footprinting vs EMSA

Footprinting shows exact binding sequences; EMSA shows whether binding occurs

Main use of EMSA

Detecting DNA–protein interactions

Competitors are always

Cold (unlabeled) and in excess

What specific competitors demonstrate

Sequence-specific protein binding

What non-specific competitors demonstrate

No binding to unrelated sequences

Antibody-induced super shift demonstrates

The DNA–protein complex contains the protein of interest

Limitation of EMSA

Cannot identify which protein binds when multiple are present

Changing competitor sequence provides

Insight into DNA sequence preferences of the protein

Alternative method to study binding

DNA footprinting (complements EMSA)