DIG Gel Shift Kit 2nd Generation Protocol & Troubleshooting

Product No. 03353591910

Protocol

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.
For further reading please also refer to attached References.

Addition of BSA
Addition of 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, the addition of BSA can improve experimental outcome.

TEN Buffer
To ensure proper annealing of single-stranded oligos they should be dissolved in TEN buffer and not in water.

Troubleshooting

Unspecific Shifts

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.

Competitor

• 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).

Addition of 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.