ECL EMSA: Problems

  
        
Problems Possible reasons Solutions
No shifted bands & no free probes are observed on x-films
Poor labeling of nucleic acid. Check labeling efficiency.
No enough amount of IR probes used. Use more IR Fluo-probes.
DNA/RNA probes are Degraded The solutions should be DNase free or RNase free.
Probes unable to bind to target proteins Label DNA/RNA with large molecules may prevent probes from binding to target proteins.
No or low binding of nucleic acid to membranes Use correct membranes with capacity binding to small Oligos (20-30bp).
Check efficiency of UV-crosslinking.
HRP or AKP inactivated or lost activity Check activity of HRP or AP.
Bad HRP/AKP Substrates Check ECL or AKP substrates. Use fresh ECL substrates.
No shifted bands & no free probe bands with imagers. Same problems as above Solutions as above
Low sensitivity of HRP/AKP Substrates Use high sensitivity substrates
Low sensitivity of imagers or scanners Use x-ray film or imager with high sensitivity.
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 requires greater than 1 h 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
Only free probe bands can be observed Proteins degraded Use high quality extraction kits with protease inhibitor.
Nuclear extraction at low temperature.
Store extracts at -80 or liquid nitrogen
Include RNase inhibitor with reaction if probe is RNA
Perform Western bot to check target protein.
Not enough proteins used Protein concentration should be 1-3 ug/ul. Total protein 2-3ug is used for EMSA.
Used Too much poly[dI:dC]   Poly[dI:dC] non-specifically bind proteins. Too much Poly[dI:dC] would reduced specific complexes.
High volume of samples are used. Nuclear proteins is extracted by buffers with high salts. Using large volume of samples increases salt concentration and reduce or block formation of DNA/RNA-protein complexes.
Only free probe bands and non-specific bands can be observed Sample without target proteins Express target protein by an external gene or change a cell line with target protein
Target proteins are inactivated Treat cells with proper cell factors or other stimulating factors
Target proteins are degraded Treat cells with proper cell factors or other stimulating factors
Free nucleic acid migrates normally. Nucleic acid mobility unchanged in presence of protein No target protein or low expression of target protein. Perform Western bot to check target protein.
Total protein concentration is low. Not enough cells/tissues are used or nuc. proteins are lost during extraction. Nuc. proteins should be >1ug/ul. Verify protein concentration. Use larger volumes of protein stock or more concentrated stock in preparing sample
Protein is inactive Run Western blot to check level of total and nuc. proteins. Activated TF would migrates into nucleus.
 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
Target proteins are decayed. Run Western blot with fresh extracts and extracts in storage.
Protein is negatively charged and comigrates with nucleic acid Check migration of protein alone under EMSA conditions. If protein comigrates with nuclei acid, reduce pH of binding and electrophoresis
buffers.
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 (approximately 50 V cm–1) until samples enter the gel, then reduce to approximately 10 V cm–1
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 polyacrylamid

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