Home \ Troubleshooting Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis

Troubleshooting Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis


This troubleshooting page gives the problem, possible cause and suggested solution for problems during the SDS-PAGE application:

Problem: Weak of missing protein bands
The protein/antigen quantity is below the detection level of the stainIncrease the sample concentration. Use a more sensitive stain.
The proteins are not fixed in the gelUse a stain which will fix the proteins. Use a gel fixing solution.
Proteins have ran off the gelUse a SDS-PAGE gel with a higher % acrylamide.
Proteins are degradedMake sure there is no protease contamination. Ensure the samples did not freeze-thaw.
The small-peptides (<4 kDa) did not fix in the gelFix the gel with 5% glutaraldehyde. Rinse the gel well with water before staining.
Problem: Poor band resolution
The concentration of the protein is too highDecrease protein concentration.
Sample volume is too largeIncrease protein concentration.
Gel concentration is not correctIf the size of the protein is unknown, use a 4%-20% gradient gel.
The gel is too oldOrder fresh precast gels or cast a fresh gel.
There is excess micelle formationDo not exceed 200 µg SDS/30 µl sample.
The run is too fast because buffers are too dilutedIncrease the buffer concentration.
The run is too fast because the current is too highDecrease the voltage by 25-50%.
The protein bands are not sufficiently resolvedInsufficient electrophoresis has taken place, prolong the run. The gels pore size is not correct for the proteins that need to be separated. Use a gel with a different % acrylamide.
Problem: Band smearing
The voltage used is too highDecrease the voltage by 25-50%.
The concentration of the protein is too highReduce the amount of protein loaded on the gel.
The salt concentration is too highDialyze the sample, precipitate the protein with trichloroacetic acid (TCA) or use a desalting column
Problem: Bands are skewed or disorted
The salt concentration is too highDialyze the sample, precipitate the protein with trichloroacetic acid (TCA) or use a desalting column.
The polymerization around the sample wells is poorIncrease the amount of ammonium persulfate and TEMED.
Excessive pressure has been applied to the gel plates when the gel is placed into the clamp assemblyThe screws on the clamp should not be too tight.
Material in the gel is insoluble or the pore size are inconsistent throughout the gelFilter the gel reagents, ensure that the gel mixture is well mixed and degassed before pouring the gel.
The gel interface is unevenWith a spirit the gel apparatus can be made even. Overlay the separating gel carefully with water.
Heating of the gel is unevenUse a cooled apparatus or reduce the current.
Problem: Running defects and gel casting
Time that the gel polymerizes is too longIncrease ammonium persulfate or TEMED or use fresh ammonium persulfate and new TEMED. The temperature is too low, cast at room temperature. Quality of the acrylamide or bis is poor. The concentration of the thiol reagent is too high which inhibits polymerization. A more rapid polymerization can be accomplished by degassing the acrylamide solution.
The gel does not polymerizeTEMED and ammonium persulfate are left out of the gel mixture. Increase ammonium persulfate or TEMED. Use fresh ammonium persulfate and new TEMED. The temperature is too low, cast at room temperature. Quality of the acrylamide or bis is poor.
The gel is too softQuality of the acrylamide or bis is poor. There is too little crosslinker, increase the amount of bisacrylamide.
The gel turns whiteThe bis concentration is too high, recheck the amount that is used
The upper buffer chamber leaksThe upper buffer chamber is overfilled. Assemble the casting apparatus correctly
There is leaking during gel castingl casting Glass plates might be chipped. The glass plate might not be aligned well. Use Vaseline at the spacers
The gel cracked during polymerizationThere is an excess of heat generation, use cooled reagents.
The samples do not sink to the bottom of the wellThere is insufficient glycerol in the sample buffer. Comb is removed before the stacking gel has properly been polymerized. Let the gel polymerize 30 minutes before removing the combs.
SDS is not added to sampleThere are no net negative charges on proteins, the protein will not move down the gel, ensure SDS has been added to the sample.
Sample preparation is yellow in colorThe solution is acidic, add NaOH until the solution turns blue. There is too little bromophenol blue in the sample buffer.
The run takes an unusual long timeThe buffers are too concentrated, dilute the buffer if necessary. The current is too low, increase the voltage.
Some bands do not move down the gelThere might be air bubbles in the gel under the affected lanes. Make sure there are no air bubbles in the gel
The gel detaches from the glass platesThe glass plates are not clean.
Gel has cracked during electrophoresisThe running conditions are too warm, it happens faster with high percentage gels.
The sample wells are poorWhen the comb is not removed carefully , the wells can be broken or distorted. When stacking gel resists the removal of the comb, use a gel with lower % acrylamide.
The base of the sample well appears to be dragged downwardsHigh molecular mass might be trapped. Check the sample for nucleic acid and remove them if they are in significant quantities.
Problem: The protein has aggregated
Proteins have aggregatedThe salt concentration is too high, precipitate and resuspend in lower salt buffer. Disulfide bonds are formed by the proteins in the complex mixture because of insufficient reducing agent. Prepare new sample buffer. Some samples aggregate on boiling, threat them at a lower temperature (60°C).
There is protein precipitation in the wellThe proteins are hydrophobic, add 4-8 M urea to the sample.
Band streakingThe sample is too concentrated or there is not enough SDS. Dilute the sample with more SDS solution.
Problem: There are artifact bands
There are fewer bands than expected and there is a heavy band at the dye frontThe gel percentage is too low, increase the % acrylamide in the gel.
There is lateral band spreadingBefore the power was turned on the sample diffused out of the well. The time between sample application and power start up should minimized.
There is vertical streaking of proteinSample precipitation. Centrifuge all the samples before they are loaded in the wells. The sample is overloaded, dilute the sample or reduce the voltage by about 25% to minimize streaking.
‘Smile effect’The center of the gel is running hotter than either ends. Decrease the power setting and check the buffers.
An approximate 67 kDa band is observed in reduced samplesThis band is from the excess of the reducing agent
(β-mercaptoethanol). By adding iodoacetamide to the equilibration buffer just before applying the sample to the gel artifact bands will be eliminated.
There are doublets observed where a single protein band is expectedA portion of the protein sample may have re-oxidized during the run, or may not have been fully reduced prior to the run. Prepare fresh sample solution using fresh β-mercaptoethanol or dithiothrietol (DTT), or increase the amount in the sample buffer.
Helpful links / references www.protocol-online.org / http://www.omx-online.com/calculator.html

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