DIG Gel Mobility Shift Assay Troubleshooting

The DIG Gel Shift Kit, 2nd generation (Product No. 03353591910) contains reagents for nonradioactive 3′-end labeling of oligonucleotides for use in a gel mobility shift assay, also referred as an electrophoretic mobility shift assay (EMSA). The kit also contains electrophoresis reagents and an enzyme-labeled antibody to facilitate the detection of DNA-protein complexes.

The DIG Gel Shift Kit uses recombinant terminal transferase and digoxigenin (DIG)-11- dideoxyuridine triphosphate (ddUTP), which makes the labeling reaction flexible. The kit can be used to label 3′ ends of any oligonucleotide, whether it has a 5′- overhanging end, a 3′-overhanging end, or blunt ends. Both single- and double-stranded DNA can be labeled.

TROUBLESHOOTING a gel mobility shift assay

Non-specific Competitor Reaction
To prevent nonspecific binding of the analyzed protein or extract to the DNA fragment or oligonucleotide, nonspecific competitor nucleic acid is added to the binding reaction. If a GC-rich binding sequence is expected, poly [d(I-C)] should be applied; if an AT-rich binding sequence is expected, poly [d(A-T)] should be applied as a nonspecific competitor.

Note: The optimum amount of competitor DNA used must be determined empirically.

Adding BSA
Adding BSA (250 μg/mL) can yield higher signals in gel shift experiments, especially when using extracts. Because extracts may contain proteases, their activity can be buffered by adding BSA. In combination with specific DNA-binding factors that require stabilization, adding BSA can improve experimental outcome.

TEN Buffer
To ensure proper annealing, dissolve single-stranded oligos in TEN buffer and not in water.

Labeling Efficiency
It is important to determine the labeling efficiency of your DIG-labeled binding oligonucleotide. Only with this information is clear that specific signals can be obtained.

Adding Competitors

  • Optimize binding by adding different concentrations of competitors (also test different competitor combinations).
  • What type of competitor are you using? This is important for determining specificity of the binding factor. Roche recommends using a nonspecific, unlabeled oligonucleotide. When using higher concentrations of this competitor produces no reduction in binding efficiency should occur when the protein specifically binds the labeled oligo sequence (it is also possible to use a mutated oligo or the control oligo from the kit).

Adding poly d(IC) or poly d(AT)
Depending on the GC content of the binding sequence, test addition of poly d(IC) or poly d(AT)–best test both. See Technical Tip (link in documents)

Salt Conditions
Salt conditions are also important (these are specific for the DNA-binding protein to be detected); please refer to p. 15 (Modification of reaction conditions / binding buffer conditions) of the Instruction Manual.

Gel mobility of DNA-protein complexes
A reason DNA/Protein complexes do not run into the gels could be the size and potential multiple binding sites of the DNA fragment. In this case, try reducing the size of the DNA fragment and/or use an oligonucleotide.

  • Use a native PAA gel, and not an SDS gel.
  • Poly d(IC) supplied with the kit prevents smear formation due to nonspecific protein binding.

Oct controls supplied with the DIG Gel Shift Kit, 2nd generation should easily run into the gel. Oct2a can be sensitive to temperature, but only slightly, meaning the gel can be run at room temperature.

Materials
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