Table 3.

Troubleshooting

Problem	Possible Causes	Possible Remedies
No bands visible after electrophoresis	Low nucleic acid concentration	Verify nucleic acid concentration. Repeat experiment with appropriate concentration of nucleic acid.
	Inefficient labeling	Check reaction components for concentration and enzyme activity
	Nucleic acid degradation	If sample is radioactive, verify that nucleic acid is intact DNA by running a sequencing gel. If nuclease activity is suspected, treat buffers with diethyl pyrocarbonate. Exclude divalent cations wherever possible. Commercially available RNase inhibitors are useful to protect RNA substrates. Commercially-available phosphatase inhibitors can prevent substrate dephosphorylation.
All bands are smeared or streaked	Uneven gel polymerization	Use fresh gel components. Degas thoroughly before polymerization. If polymerization interfered with casting gel, reduce TEMED concentration. If gel required >1h to polymerize, increase ammonium persulfate concentration.
	Excessive gel heating	Check concentrations of gel and running buffer. If they are correct, reduce voltage during electrophoresis.
	Sample conductivity too high.	Reduce salt concentration in nucleic acid or sample buffer.
Free nucleic acid migrates normally. Nucleic acid mobility unchanged in presence of protein	Low protein concentration	Verify protein concentration. Use larger volumes of protein stock or more concentrated stock in preparing EMSA samples.
	Protein is inactive	Run SDS-polyacrylamide gel electrophoresis to rule out degradation. If the binding protein is an enzyme, test for activity. Test higher concentrations of protein to detect residual binding activity. A new, more active preparation of protein may be necessary.
	Protein is negatively charged and co-migrates with nucleic acid	Check migration of protein alone under EMSA conditions. If protein co-migrates with nucleic acid, reduce pH of binding and electrophoresis buffers 64.
Free nucleic acid migrates normally. No nucleic acid detectable in samples containing protein.	Nucleic acid degradation	Verify that nucleic acid is intact. If nuclease activity is suspected, treat glassware and buffers with diethyl pyrocarbonate. Exclude divalent cations wherever possible. Use commercial RNase and phosphatase inhibitors.
	Binding occurred but complexes dissociated during electrophoresis. Liberated nucleic acid is too diffuse to detect.	Minimize gel running times; use more concentrated gel; include stabilizing solutes in gel buffer; reduce salt concentration in binding and electrophoresis buffers to increase electrostatic stabilization; lower electrophoresis temperature (run gel in cold room); reduce or eliminate competing nucleic acid (this can be added back with care once a useful binding signal is obtained).
Free nucleic acid migrates normally. Complex bands smeared or streaky	Binding occurred but complexes dissociated in gel during electrophoresis.	Minimize gel running times; use more concentrated gel; include stabilizing solutes in gel buffer; reduce salt concentration in binding and electrophoresis buffers to increase electrostatic stabilization; lower electrophoresis temperature (run gel in cold room); reduce or eliminate competing nucleic acid.
	Binding occurred but complexes dissociated in the well prior to electrophoresis.	Minimize interval that sample is in well before electrophoresis. Complexes may be destabilized by component(s) of running buffer. If so they may be more stable in gel and running buffers that more closely resemble the composition of binding buffer.
	Binding occurred but complexes dissociated in the well during electrophoresis.	To minimize gel “dead time”, use smallest sample volume possible; conduct electrophoresis at high voltage (~50 V/cm) until samples enter the gel, then reduce to ~10V/cm.
	Binding occurred but samples containing protein are too salty.	Reduce salt concentration in protein stock and/or in binding buffer.
Free band is sharp, complex band(s) are broad and indistinct	Heterogeneous protein.	Multiple species may be due to post-translational modification or to partial degradation without loss of binding activity.
Complex and free bands are broad and indistinct	Sample zone is too large (measured from top of sample to bottom of well) at the start of electrophoresis	Reduce sample volume. Increase density of sample (e.g., increase glycerol concentration) to facilitate gel loading. Minimize time between loading and electrophoresis
	Electrophoresis period too long	Reduce run-time
	Nucleic acid degradation	Verify that nucleic acid is intact. If nuclease activity is suspected, treat extracts and buffers with diethyl pyrocarbonate. Exclude divalent cations wherever possible. Use RNase and phosphatase inhibitors.
Nucleic acid stuck in well, no free species visible	Protein/nucleic acid ratio is too high	Reduce the concentration of protein or increase the concentration of unlabeled nonspecific competitor
	Protein is aggregated	Change binding conditions to improve protein solubility. Possible modifications: add solutes that stabilize folded (compact) forms of proteins (e.g., glycerol); keep protein stocks and binding reactions at ice temperature; avoid freeze-thaw cycles with protein stocks; include non-ionic detergents in protein storage buffer and/or binding buffer
	Free nucleic acid and complexes are too large for gel system	Try lower percentage polyacrylamide or reduce the acrylamide/bisacrylamide ratio. Test agarose gel as alternative to polyacrylamide.