Troubleshooting Hints and Tips for NIR Fluorescent EMSAs

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.

No shift bands detected or weak signal

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.

Spots or speckling

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