Gel shift assays, or electrophoretic mobility shift assays (EMSA), provide a simple method to study DNA-protein interactions. Existing mobility shift assay protocols can be easily converted to infrared fluorescent assays by replacing the existing DNA oligonucleotides with IRDye® infrared dye end-labeled oligonucleotides. Binding and electrophoresis conditions are the same as any other EMSA detection method.
A DNA oligonucleotide end-labeled with IRDye 700 infrared dye is a good substrate for protein binding. Using the Odyssey Infrared Imaging System, IRDye infrared dye labeled DNA detection is linear within a 50-fold dilution range from 9.1 fmol to 0.18 fmol. Infrared assays can be completed in less than two hours with no gel transfer or film exposure. The gel doesn’t even have to be removed from the glass plates for imaging. Plus, the gel can be placed back into the electrophoresis unit and run longer, if necessary.
Advantages of Infrared:
| Infrared |
Radioisotope |
| Easy access and disposal |
Regulatory procedures, disposal limitations |
| Dye is stable for a long time |
Short half-life of the label |
| Non-hazardous |
Hazardous |
| Gel (glass plates) can be easily imaged on the Odyssey |
Lengthy incubations with autoradiographic film |
| Detection of the probe is rapid |
Time-consuming and inconvenient |
| Gel can be replaced back and run longer |
Not possible |
Available Sizes:
| Description |
Cat. No. |
| EMSA Buffer Kit for the Odyssey |
LIC-829-07910 |
| EMSA Oligo, STAT3 (IRDye 700)* |
LIC-829-07922 |
| EMSA Oligo, CREB (IRDye 700)* |
LIC-829-07923 |
| EMSA Oligo, NFkB (IRDye 700) |
LIC-829-07924 |
| EMSA Oligo, AP-1 (IRDye 700) |
LIC-829-07925 |
| EMSA Oligo, Sp-1 (IRDye 700) |
LIC-829-07926 |
| EMSA Oligo, E2F (IRDye 700)* |
LIC-829-07927 |
| EMSA Oligo, HIF-1 (IRDye 700) |
LIC-829-07929 |
| EMSA Oligo, OCT-1 (IRDye 700)* |
LIC-829-07930 |
| EMSA Oligo, PAX-5 (IRDye 700)* |
LIC-829-07931 |
| EMSA Oligo, ARE (Androgen Receptor) (IRDye 700)* |
LIC-829-07933 |
IRDye 700 infrared dye-labeled EMSA oligonucleotides are supplied as 25 μl of 50 nM
double-stranded DNA.
Please note: Oligos with an * require up to two weeks for processing and delivery. |
| Description |
Cat. No. |
| EMSA Buffer Kit for the Odyssey |
LIC-829-07910 |
Tube |
Reagent (Reagents for 100 EMSA Reactions) |
Volume |
1 |
10 X Binding Buffer (100mM Tris, 500mM KCl, 10mM DTT; pH 7.5) |
500ul |
2 |
25mM DTT/ 2.5% Tween-20 |
500ul |
3 |
Poly (dI-dC) (1ug/ul) in 10mM Tris, 1 mM EDTA, pH 7.5 |
125ul |
4 |
Sheared Salmon Sperm DNA (0.5 ug/ul) in 10mM Tris, 1 mM EDTA, pH 7.5 |
125ul |
5 |
50% Glycerol |
500ul |
6 |
1% NP-40 |
500ul |
7 |
1M KCl |
500ul |
8 |
100mM MgCl2 |
500ul |
9 |
200mM EDTA, pH 8 |
500ul |
10 |
10X Orange Loading Dye |
500ul |
| Storage: -20°C |
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