Figure 1. AP-1 EMSA using IRDye 700 AP-1 oligonucleotide duplex. Nuclear extracts of HeLa, or HeLa treated with serum, were used to visualize increased AP-1 binding as a result of serum treatment. Competition reactions contained 100-fold molar excess of unlabeled oligo duplex. Image captured with Odyssey Classic Imager. Arrow indicates mobility shift of fluorescent oligos. Asterisk indicates reduction of mobility shift when an excess of unlabeled competitor DNA is added.
Because precise binding conditions are specific to each protein:DNA pair, universally appropriate binding conditions cannot be recommended. The user should establish binding reaction conditions for each protein:DNA pair. Binding buffers for this method are very similar to other mobility shift detection methods. Optimized binding conditions are provided for all IRDye® 700 EMSA oligonucleotides.
The Odyssey® EMSA Buffer Kit (P/N 829-07910) simplifies optimization of binding reactions. The kit provides a general binding buffer, various supplementary components, 10X Orange loading dye, and a protocol for optimization.
After the addition of DNA to the protein-buffer mix, reactions are incubated to allow protein to bind to DNA. Time required for binding is the same as when radioactively-labeled DNA fragments are used; typically 20-30 minutes at room temperature. Since IRDye reagents are sensitive to light, protect binding reactions from light during incubation (e.g., put tubes in a drawer or cover the tube rack with aluminum foil). After incubation, native loading dye is added to the binding reaction.
IMPORTANT: It is critical to avoid all blue loading dyes (e.g., bromophenol blue), because they will be visible on the Odyssey image. Use 10X Orange loading dye instead (P/N 927-10100).
NOTE: In some cases, DNA control reactions (no protein) may have lower signal than reactions containing protein. This may be due to lower stability of the dye in certain buffer conditions. Addition of 5 mM DTT and 0.5% Tween 20 to all reactions reduces this phenomenon.
Competitor DNAs are used to confirm specificity of protein:DNA binding.
Specific competitors contain exactly the same consensus sequence as the labeled probe. The competitor is unlabeled, and is added to the binding reaction in a large molar excess (~200-fold). Unlabeled competitor DNA out-competes the labeled probe for binding to the protein, eliminating or reducing the mobility shift.
Mutant competitor DNAs are also used to determine binding specificity, by competing with the wild-type binding sequence. Specific binding is confirmed if the mutant DNA (which is typically unlabeled) does not reduce the binding of labeled wild-type DNA.
Two-color competition experiments can be performed with mutant competitors, if desired. The wild-type oligos are end-labeled with IRDye 700, and mutant oligos with IRDye 800. Two-color imaging of mutant vs. wild-type binding is then performed with the Odyssey Imager. In Fig. 2, non-specific binding of the mutant oligos is very intense (800 nm image, green); however, there is no decrease in wild-type binding (700 nm image, red).
Figure 2. Two-color analysis of AP-1 binding specificity. HeLa cells were treated with serum for 4 h. Nuclear extracts (2.5 μg) were used for EMSA analysis with IRDye 700 AP-1 consensus oligonucleotide duplex (red). A mutant AP-1 competitor oligo duplex labeled with IRDye 800 (green) was also added, at increasing ratios relative to the AP-1 consensus oligos. Ratios ranged from 1-fold to 5-fold molar excess of mutant competitor. Addition of mutant oligos did not affect the mobility shift of the AP-1 consensus oligos. Wet gel was imaged with Odyssey® Classic Imager.