IRDye® 700 oligos include an optimized protocol to measure the protein:DNA interaction. See the specific EMSA oligo pack insert for more information.
Native pre-cast polyacrylamide gels such as 5% TBE (BioRad) or 4-12% TBE (Invitrogen) are recommended. Alternatively, the recipe below can be used to prepare a 4% native gel.
NOTE: The protein shift detected on each gel type (i.e., 5% vs 4-12%) will be unique.
Prepare 4% native polyacrylamide gel containing 50 mM Tris, pH 7.5; 0.38 M glycine; and 2 mM EDTA:
Pour the gel between glass plates and wait about 1-2 hours to polymerize.
EMSA oligonucleotides from LI-COR Biosciences are pre-annealed.
For NFκB IRDye 700 oligonucleotide, the following binding reaction is a good starting point.
|10X Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT; pH 7.5||2|
|Poly(dI•dC) 1 μg/μL in 10 mM Tris, 1 mM EDTA; pH 7.5||1|
|25 mM DTT/2.5% Tween®20||2|
|IRDye 700 NFκB||1|
|Raji nuclear extract (Positive control) (5 μg/μL)||1|
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature. Since IRDye 700 infrared dye is sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put tubes into a drawer or cover the tube rack with aluminum foil).
NOTE: For best results, electrophoresis should be performed in the dark (simply put a cardboard box over the electrophoresis apparatus).
Gels can be imaged either inside the glass plates or removed from the plates. When removing gel from the glass plates, take care not to deform or tear the gel. Scan the gel. Please refer to your manual for specific information on your model of imager.
One of the benefits of Odyssey EMSA analysis is image quantification. However, there are several issues to consider when using the Odyssey Imager to quantify EMSA results.
Figure 1. IRDye 700 NFκB oligonucleotides were separated on a native polyacrylamide gel (4-12% TBE, Invitrogen EC62352BOX) and imaged on the Odyssey Infrared Imaging System.
Figure 2. The uppermost shifted band in Lanes 2-7 of Figure 1 was analyzed to determine the level of NFκB binding to the IRDye 700 NFκB oligonucleotides.