Download Mobility Shift Assay

Mobility Shift Assay
Protein bound to a small piece of DNA will alter the electrophoretic mobility of that DNA fragment. This
allows the analysis of protein-DNA interactions, including the measurement of binding rates, affinity, and
specificity. In addition, bound and unbound DNA may be isolated from the gel and used for further types of
analysis such as methylation interference or uracil interference.
In the mobility-shift (or gel-shift) assay, end labeled DNA is allowed to bind protein. The resulting DNA
protein complexes are then run on a non-denaturing PAGE gel and the gel is dried and autoradiographed.
The buffer composition of the PAGE gel is varied from the standard TBE gel, because lower ionic strength
is needed to facilitate the DNA protein binding. It is interesting to note that binding interactions which can
dissociate in free solution within 1 minute show altered mobility on gels which require more than an hour to
run. It is theorized that the gel matrix forms a "cage" around the DNA-protein complex, which prevents the
components from diffusing away from each other, thus promoting re-association and in effect stabilizing the
complex. This would argue that the ratio of bound and unbound material seen on the gel is a direct measure
of the fraction of bound material in the sample as it entered the gel. Thus, the conditions set in the binding
reaction are of critical importance.
Mobility Shift Assay
PROBE PREPARATION:
A probe of the proper size is cut from 10 µg of plasmid clone, using restriction enzymes which will yield
probe of 50-150 bp, with one 5' overhanging end.
Label the probe with 32P dNTP and Klenow fragment, to fill in the overhang. To the restriction digest
reaction, add 100µCi of 32P dNTP (chosen to be part of the filled in region) and 0.2 mM of the other
necessary dNTP's.
Add 3 units of Klenow fragment and incubate 30 minutes at room temperature.
Add an additional aliquot of a mixture of all 4 dNTP's to a final concentration of 0.2mM, and incubate
another 10 minutes.
Add 0.1 volume 3M Sodium Acetate, 3 volumes Ethanol, precipitate the DNA and wash once with 70%
Ethanol to remove the bulk of the unincorporated label.
Isolate the probe on an Agarose gel by electrophoresis onto a DEAE membrane.
Probe should be labeled to 107 - 108 cpm/µg.
BINDING REACTION:
Mix:
104 cpm of DNA probe
2µg nonspecific DNA (calf thymus DNA, or synthetic polymer)
20 µg sample protein
1 µl glycerol in a final volume of 15 µl
Incubate at 30°C for 15-30 minutes.
This gives a basic framework for a binding reaction. Components which may be needed to ensure binding
include glycerol (10%), KCl (50mM) and DTT (1mM). Buffer conditions must be optimized for each
protein/DNA combination studied.
ELECTROPHORESIS:
The mobility shift assay gel uses a low ionic strength buffer system, to avoid salt effects on binding
constants. This necessitates buffer circulation between upper and lower chambers to prevent buffer
exhaustion and pH shifts during the run.
10X Gel Buffer:
67mM Tris HCl pH 8.0
33mM Sodium Acetate
10mM EDTA
Mix components and adjust pH to 8.0
Use this buffer at 1X for upper and lower buffer chambers.
Gel Mix:
6.7ml 30% Acrylamide (AcrylaGel )
1.25ml 2% Methylene Bis-Acrylamide (Bis-AcrylaGel)
1.25ml Glycerol
36ml H2O
5 ml 10X gel buffer
Just prior to pouring, add 125µl 10% APS and 45µl TEMED to initiate polymerization.
Gel assembly:
Cast the gel in a standard format (16cm plate) cassette, with 1.5mm spacers and wells at least 0.8cm wide.
Pour using standard techniques, and allow to polymerize one hour.
Run Conditions:
Recirculate the buffer at least 10ml/min.
Pre-run gel for 1.5 hours at 6V/cm
Preload at least one well with 0.01% Bromophenol Blue in 10% glycerol, to provide a tracking dye. No dyes
are included in the binding reactions.
Load binding reactions and run gel at approximately 12 V/cm for 1-2 hours. Adjust the run voltage so that
plates do not become warm, because increases in temperature will alter binding equilibria. A current of 2530 mA is sufficient for a 16 cm gel. Gels may be run at higher voltage in a cold room.
RESULTS:
Bound DNA will appear as a more slowly migrating band, which is not visible in the lanes without protein.
The band will disappear on addition of unlabeled competitive DNA sequences.
NEXT TOPIC: DNA/RNA Purification from PAGE Gels
Products Related to this Discussion:
AcrylaGel
Ready-to-use, 30% acrylamide solution in distilled/deionized water. 18 megOhm water. 0.2 micron
filtration.
Bis-AcrylaGel
Ready-to-use, 2% acrylamide solution in distilled/deionized water.
Tris - ULTRA PURE
Purified to remove ammonia and amine contaminants.
Ammonium Persulfate - ULTRA PURE
Exceeds ACS standards. Low absorbed water results in consistent initiation.
TEMED - ULTRA PURE
Fractionally distilled to remove all trace metals and amine impurities. Stored under nitrogen.
EDTA - ULTRA PURE
Chelating agent added to electrophoresis buffers.
Glycerol - ULTRA PURE
Conforms to the stringent requirements. Molecular biology