Troubleshooting for EMSA/Gel Shift Assays

Weak or no signal

Possible Cause	Possible Solution
Did not add DTT/Tween^® 20 to binding reaction	Add 1 µL of 25 nM DTT/2.5% Tween 20 to binding reaction
Not enough IRDye^® labeled DNA used	Increase amount of IRDye labeled DNA added to the reaction
Target DNA degraded	Verify integrity of DNA
Imaged in the wrong channel of the Odyssey^® Imager	When using IRDye 700-labeled DNA, turn on the 700 nm laser.

Possible Cause	Possible Solution
Used incorrect scan intensity setting	Scan the gel again using a Scan Intensity of 8 in the Odyssey Application software, or use the AutoScan mode in the Image Studio™ software
Incorrect focus offset	Adjust the Focus Offset in the Odyssey software or Image Studio software to equal the thickness of the glass plate plus half the thickness of the gel, and scan again.
DNA:protein complex disrupted due to heat or vortexing	Run gel with cooled buffer. Do not vortex binding reaction.
Not enough extract	Add more extract to reaction.
Degraded extract	Minimize freeze/thaw cycles. Use protease inhibitors.
EMSA binding reaction not fully optimized	Use the additives in the Odyssey EMSA Buffer Kit (P/N 829-07910) to optimize the binding reaction conditions.

Possible Cause	Possible Solution
Contamination on glass surfaces	Clean glass gel plates and the Odyssey scanning surface with isopropanol